Combination therapy intravaginal rings

ABSTRACT

The present invention provides improved intravaginal drug delivery devices, i.e., intravaginal rings, useful for the prophylactic administration of dapivirine in combination with either an antimicrobial compound or a contraceptive to a human. The present invention also provides methods of blocking DNA polymerization by an HIV reverse transcriptase enzyme, methods of preventing HIV infection in a female human, methods of treating HIV infection in a female human, methods of preventing unintended pregnancy in a female human, and methods of preparing intravaginal rings.

RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 16/155,473, filed Oct. 9, 2018, which is adivisional of U.S. patent application Ser. No. 14/540,530, filed Nov.13, 2014, which, in turn, claims priority to U.S. ProvisionalApplication No. 61/904,073, filed Nov. 14, 2013. The entire contents ofeach of the foregoing applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The AIDS epidemic continues to exact a devastating toll on the health,economic and political infrastructure, and social fabric of communitiesworldwide. During 2011, almost 2.5 million people became newly infectedwith HIV bringing the total number of people living with HIV to anestimated 34.0 million. In the same year, almost 1.7 million people diedfrom AIDS, raising the global death toll to over 35 million since thefirst cases of AIDS were identified in 1981 (UNAIDS and WHO 2011 AIDSEpidemic Update; December 2011). HIV/AIDS has become the fourth biggestcause of global mortality. Over 95 percent of new infections areoccurring in developing countries, where increasing numbers of new HIVinfections threaten the sustainability of expanded access to HIVtreatment. Developing safe and effective HIV prevention technologiesthat can be made easily accessible in developing countries is, thus, anurgent public health priority.

Epidemiologic data published in the latest UNAIDS report show that womenand girls bear a severe and increasingly heavy burden of the HIVepidemic. In Eastern Europe and Central Asia, an estimated 26% of adultsliving with HIV in 2007 were women aged 15 years or older, compared with23% in 2001. In sub-Saharan Africa, women comprised 61% of HIV-infectedadults, and among young people (aged 15-24 years) the ratio of infectionhad risen to three women for every man (UNAIDS and WHO 2007 AIDSEpidemic Update; December 2007).

Each year, there are also 80 million unintended pregnancies, and 358,000women die annually from pregnancy-related complications. More than 200million women in developing countries lack access to contraception.Accordingly, there is a strong need for developing combination productsthat can prevent both HIV infection and unintended pregnancy in females.

Unprotected heterosexual intercourse is currently the leading mode ofHIV infection and unintended pregnancy among females. Correct andconsistent use of latex condoms is one proven method of preventing HIVtransmission and unintended pregnancy; however, condoms are widelyregarded as inadequate prevention options for women if they are unableto negotiate condom use for fear of abuse or accusations of infidelity.Additionally, women who have sex with men in exchange for gifts or moneymay be reluctant to use condoms if the men are willing to pay more forsex without a condom. The female condom has been marketed as analternative barrier method, but this device is relatively costly andrequires a certain level of skill, as well as acceptance by the malepartner. Developing HIV and pregnancy prevention options that women canuse with or without their partner's knowledge is a pressing globalconcern, especially given the rapidly growing HIV infection rate amongwomen and the absence of an effective vaccine.

Topical microbicides that can be self-administered to the vagina are onesuch promising alternative. Multiple clinical trials with variousmicrobicides have been completed or are currently underway, most ofwhich involve microbicides in gel formulation delivered via a single useapplicator used prior to coitus. In order for a microbicide to beeffective, it is essential that it be used correctly. Therefore it isimportant that a microbicide is acceptable to users, and it is likelythat products that can be used less frequently will be more acceptableand will achieve better user adherence. Additionally, combinationproducts that could prevent both HIV transmission and unintendedpregnancy would be more acceptable to users.

Vaginal rings that need only be replaced at relatively long intervalsmay therefore have benefits over other dosage forms that must be usedmore frequently. Ring-shaped devices for the controlled administrationof steroid substances (substantially water-insoluble drugs) into thevagina are known in the art, such as Estring®, Femring® and Nuvaring®.There are two basic types of vaginal rings: reservoir rings and matrixrings.

A reservoir ring comprises a full or partial-length core loaded with thedrug substance, which is completely surrounded by a non-medicatedsheath. Accordingly, the release of drug substances from such rings isdependent upon permeation (i.e., molecular dissolution and subsequentdiffusion) of the core-loaded drug substance through the outer sheath.Release rates can be modified by changing the thickness of therate-controlling sheath and/or the length of the drug-loaded core.Reservoir rings were developed to provide controlled (that is, constantdaily) release rates. The polymeric materials used in the constructionof commercial vaginal rings are typically hydrophobic materials, such assilicone elastomer and poly(ethylene-co-vinyl acetate) (PEVA) materials.Alternatively, polyurethane may be used.

To date, no combination therapy intravaginal rings have beensuccessfully developed. Combining different drugs in the sameintravaginal ring poses unique challenges, due to the differentsolubility and target release rates of different drugs. Accordingly,there remains a need for the development of improved intravaginal ringswhich can be loaded with a combination of antimicrobial drugs to preventthe transmission of HIV, or a combination of an antimicrobial drug and acontraceptive to prevent the transmission of HIV and unintendedpregnancy.

SUMMARY OF THE INVENTION

The present invention provides combination therapy intravaginal drugdelivery devices, i.e., intravaginal rings, useful for theadministration of therapeutic and/or prophylactic agents to a human. Theintravaginal rings of the invention may provide long-term controlledrelease of dapivirine and an antimicrobial agent, such as maraviroc,DS003, darunavir, GSK1265744 or BMS-663068, or dapivirine and acontraceptive, such as levonorgestrel, estradiol, etonorgestrel ornestorone. Surprisingly, the combination of dapivirine and either anantimicrobial compound or a contraceptive in the intravaginal rings ofthe invention results in the formation of a eutectic composition andprovides increased release rates as compared to previous intravaginalrings.

In one aspect, the invention provides an intravaginal ring comprisingdapivirine and an antimicrobial compound, wherein less than about 7 mgof dapivirine is released in vitro from said ring during an initial 24hour period of release, and wherein the release rate of theantimicrobial compound from the intravaginal ring in vitro is increasedas compared to the release rate of the antimicrobial compound from anintravaginal ring comprising the antimicrobial compound withoutdapivirine.

In another aspect, the invention provides an intravaginal ringcomprising dapivirine and a contraceptive, wherein less than about 7 mgof dapivirine is released in vitro from said ring during an initial 24hour period of release, and wherein the release rate of thecontraceptive from the intravaginal ring in vitro is increased ascompared to the release rate of the contraceptive from an intravaginalring comprising the contraceptive without dapivirine.

In another aspect, the invention provides an intravaginal ringcomprising dapivirine and an antimicrobial compound, wherein less thanabout 7 mg of dapivirine is released in vitro from said ring during aninitial 24 hour period of release, and wherein the dapivirine in theintravaginal ring increases the solubility of the antimicrobial compoundas compared to the solubility of the antimicrobial compound in anintravaginal ring comprising the antimicrobial compound withoutdapivirine.

In another aspect, the invention provides an intravaginal ringcomprising dapivirine and a contraceptive, wherein less than about 7 mgof dapivirine is released in vitro from said ring during an initial 24hour period of release, and wherein the dapivirine in the intravaginalring increases the solubility of the contraceptive as compared to thesolubility of the contraceptive in an intravaginal ring comprising thecontraceptive without dapivirine.

In another aspect, the invention provides an intravaginal ringcomprising a eutectic composition comprising dapivirine and anantimicrobial compound, wherein less than about 7 mg of dapivirine isreleased in vitro from said ring during an initial 24 hour period ofrelease.

In another aspect, the invention provides an intravaginal ringcomprising a eutectic composition comprising dapivirine and acontraceptive, wherein less than about 7 mg of dapivirine is released invitro from said ring during an initial 24 hour period of release.

In one embodiment, between about 100 and about 700 μg of dapivirine isreleased in vitro each day for 23 days after an initial 7 day period ofrelease.

In one embodiment, the antimicrobial compound is released in vitro at arate of between about 200 μg per day and about 2000 μg per day, betweenabout 400 μg per day and about 4000 μg per day, between about 550 μg perday and about 5500 μg per day, or between about 800 μg per day and about8000 μg per day for 23 days or 53 days after an initial 7 day period ofrelease.

In one embodiment, the intravaginal ring is a matrix-type ring, andwherein the contraceptive is released in vitro at a rate of about 35 μgper day to about 70 μg per day for about 53 days after an initial 7 dayperiod of release. In another embodiment, the contraceptive is releasedin vitro at a rate of about 35 μg per day to about 70 μg per day forabout 53 days after an initial 7 day period of release.

In one embodiment, the intravaginal ring is a reservoir-type ring, andwherein less than about 2 mg of the contraceptive is released from thering during an initial 24 hour period of release, and wherein about 20μg per day to about 290 μg per day of the contraceptive is released invitro after an initial 7 day period of release. In another embodiment,less than about 1 mg of the contraceptive is released in vitro from thering during the initial 24 hour period of release. In anotherembodiment, about 35 μg per day to about 70 μg per day of thecontraceptive is released in vitro after the initial 7 day period ofrelease for about 23 days. In another embodiment, about 35 μg per day toabout 70 μg per day of the contraceptive is released in vitro after theinitial 7 day period of release for about 53 days.

In one embodiment, less than about 100 μg per day of the contraceptiveis released in vitro after the initial 7 day period of release for about23 days. In another embodiment, less than about 100 μg per day of thecontraceptive is released in vitro after the initial 7 day period ofrelease for about 53 days.

In one embodiment, less than about 15 μg per day of the contraceptive isreleased in vitro after the initial 7 day period of release for about 23days. In another embodiment, less than about 15 μg per day of thecontraceptive is released in vitro after the initial 7 day period ofrelease for about 53 days.

In one embodiment, the ring is a matrix-type ring. In anotherembodiment, the ring is a platinum-catalyzed ring. In anotherembodiment, the ring comprises a silicone polymer, an EVA polymer, or apolyurethane polymer.

In another embodiment, the ring is a reservoir-type ring comprising acore and a sheath. In one embodiment, the dapivirine and thecontraceptive, or the dapivirine and the antimicrobial compound arepresent in the core of the reservoir-type ring, and the sheath is blank.In another embodiment, the dapivirine and the contraceptive, or thedapivirine and the antimicrobial compound are present in two separatehalf-length cores, and the sheath is blank. In one embodiment, thecontraceptive or the antimicrobial compound are present in the core, andthe dapivirine is present in the sheath. In one embodiment, the core isplatinum-catalyzed. In one embodiment, the core comprises a siliconepolymer, an EVA polymer, or a polyurethane polymer.

In one embodiment, the antimicrobial compound is maraviroc. In anotherembodiment, the antimicrobial compound is DS003. In another embodiment,the antimicrobial compound is darunavir, GSK1265744 or BMS-663068.

In one embodiment, the contraceptive is levonorgestrel. In anotherembodiment, the contraceptive is estradiol, etonorgestrel or nestorone.

In one embodiment, dapivirine is present in the ring in atherapeutically effective amount. In another embodiment, dapivirine ispresent in the ring in a prophylactically effective amount. In oneembodiment, about 10 to about 800 mg of dapivirine is present in thering. In one embodiment, about 100 mg of dapivirine is present in thering. In another embodiment, about 150 mg of dapivirine is present inthe ring. In another embodiment, about 200 mg of dapivirine is presentin the ring. In another embodiment, about 10 to about 30 mg ofdapivirine is present in the ring. In another embodiment, about 25 mg ofdapivirine is present in the ring. In another embodiment, about 15 mg ofdapivirine is present in the ring.

In one embodiment, the antimicrobial compound is present in the ring ina therapeutically effective amount. In another embodiment, theantimicrobial compound is present in the ring in a prophylacticallyeffective amount. In another embodiment, about 100 to about 1600 mg ofthe antimicrobial compound is present in the ring. In anotherembodiment, about 100 to about 800 mg of the antimicrobial compound ispresent in the ring. In another embodiment, about 100 mg, 400 mg, 800 mgor 1600 mg of the antimicrobial compound is present in the ring.

In one embodiment, the contraceptive is present in the ring in atherapeutically effective amount. In another embodiment, thecontraceptive is present in the ring in a prophylactically effectiveamount. In one embodiment, about 10 to about 800 mg of the contraceptiveis present in the ring. In another embodiment, about 10 to about 100 mgof the contraceptive is present in the ring. In one embodiment, about 16mg of the contraceptive is present in the ring. In another embodiment,about 25 mg of the contraceptive is present in the ring. In anotherembodiment, about 32 mg of the contraceptive is present in the ring. Inanother embodiment, about 50 mg of the contraceptive is present in thering.

In one embodiment, release rates are stable following 3 months ofstorage. In another embodiment, release rates are stable following 6months of storage. In another embodiment, release rates are stablefollowing 12 months of storage. In another embodiment, release rates arestable following 36 months of storage.

In one embodiment, the intravaginal ring has an outer diameter of about58 mm, an internal diameter of about 43 mm and a cross-sectionaldiameter of about 7.6 mm. In another embodiment, the intravaginal ringhas an outer diameter of about 57 mm and a cross-sectional diameter ofabout 7.8 mm.

In another aspect, the invention provides a method of blocking DNApolymerization by an HIV reverse transcriptase enzyme in a female humanby inserting an intravaginal ring of the invention into the vagina ofthe female human.

In another aspect, the invention provides a method of preventing HIVinfection in a female human, comprising the step of inserting anintravaginal ring of the invention into the vagina of the female human.

In another aspect, the invention provides a method of treating HIVinfection in a female human, comprising the step of inserting anintravaginal ring of the invention into the vagina of the female human.

In another aspect, the invention provides a method of preventingpregnancy and blocking DNA polymerization by an HIV reversetranscriptase enzyme in a female human by inserting an intravaginal ringof the invention into the vagina of the female human.

In another aspect, the invention provides a method of preventingpregnancy and preventing HIV infection in a female human by inserting anintravaginal ring of the invention into the vagina of the female human.

In another aspect, the invention provides a method of preventingpregnancy treating HIV infection in a female human by inserting anintravaginal ring of the invention into the vagina of the female human.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B depict the mean daily (FIG. 1A, ±sd) and cumulative(FIG. 1B) release of dapivirine (DAP) from MED-4870 matrix ringscomprising DAP (100, 150 and 200 mg per ring), with or withoutlevonorgestrel (LNG) (0, 16 and 32 mg per ring) over 60 days.

FIG. 2A and FIG. 2B depict the mean daily (FIG. 2A, ±sd) and cumulative(FIG. 2B) release of LNG from MED-4870 matrix rings comprising LNG (16and 32 mg per ring), with or without DAP (0, 100, 150 and 200 mg perring) over 60 days.

FIG. 3A and FIG. 3B depict the mean daily (FIG. 3A, ±sd) and cumulative(FIG. 3B) release of DAP from MED-4870 matrix rings comprising DAP (200mg per ring), with or without LNG (32 mg per ring) over 92 days.

FIG. 4A and FIG. 4B depict the mean daily (FIG. 4A, ±sd) and cumulative(FIG. 4B) release of LNG from MED-4870 matrix rings comprising LNG (32mg per ring) and DAP (200 mg per ring) over 92 days.

FIG. 5A and FIG. 5B depict LNG (FIG. 5A) and DAP (FIG. 5B) cumulativerelease versus time plots for various ring formulations described inExample 1.

FIG. 6A and FIG. 6B depict the mean daily (FIG. 6A, ±sd) and cumulative(FIG. 6B) release of DAP from rings comprising a LNG-loaded core (1%w/w, DDU-4320) (sets A and C) or MED8-6382 (set B) and a DAP-loadedsheath (0.3125% w/w, DDU-4320) (n=6). The DAP daily release plots arevery similar for the three ring sets. Cumulative release plots show aslight increase for Set B, likely due to the release enhancing influenceof propanol produced as a by-product of the curing of the MED8-6382silicone system.

FIG. 7A and FIG. 7B depict the mean daily (FIG. 7A, ±sd) and cumulative(FIG. 7B) release of LNG from rings comprising a LNG-loaded core (1%w/w, DDU-4320) (sets A and C) or MED8-6382 (set B) and a DAP-loadedsheath (0.3125% w/w, DDU-4320) (n=6).

FIG. 8 is a table depicting a summary of the DAP release data for eachring set in Example 2. Release of DAP follows t^(0.5) kinetics, i.e.daily release declines over time. This is typical of matrix rings, wheredrug is dispersed uniformly throughout the silicone elastomer. It istherefore not appropriate to report the average daily release rate ofDAP in terms of μg/day, since this would be misleading. According toHiguchi's equation, cumulative release from matrix devices is directlyproportional to the square root of time. The value in the table wasobtained by plotting cumulative release against the square root of timeand determining the gradient of the best-fit linear trend line. Linearregression analysis refers to the linear regression of a plot ofcumulative release against root time.

FIG. 9 is a table depicting a summary of the LNG release data for eachring set in Example 2. Release of LN follows zero order kinetics, i.e.the mass released each day is constant. This is because LN is loadedinto the core of the ring and is surrounded by a rate-limiting siliconeelastomer sheath (reservoir ring). It is therefore appropriate to reportthe mean daily release of LN in terms of μg/day. The value in the tablewas obtained by plotting cumulative release against time and determiningthe gradient of the best-fit linear trend line. Linear regressionanalysis refers to the linear regression of a plot of cumulative releaseagainst root time.

FIG. 10 depicts details of the ring configurations used in Example 3.Configuration 1 depicts the visible gap between the two ends of thecore, due to the cut made in the core prior to over molding. The twoseparate half-length cores are clearly visible in the Core 2 imagewherein the white core is the DAP-loaded segment (white appearance dueto the micronized DAP), while the more transparent core is theLNG-loaded segment (LNG was not micronized; small particles of LNG wereclearly visible in the silicone elastomer, although these may not beevident from the image in the table.)

FIG. 11A and FIG. 11B depict the mean daily FIG. 11A, ±sd) andcumulative (FIG. 11B) release of dapivirine from each ring set (n=6).

FIG. 12A and FIG. 12B depict the mean daily (FIG. 12A, ±sd) andcumulative (FIG. 12B) release of LNG from each ring set (n=6).

FIG. 13 depicts the four types of ring configurations used in Example 4.Ring type C1 is a matrix-type ring, comprising both dapivirine and LNG.Ring type C2 is a reservoir-type ring, with both dapirivine and LNGloaded in a core which is surrounded by a blank sheath. Ring type C3 isa reservoir-type ring, with dapivirine loaded in half of the core andLNG loaded in the other half of the core, surrounded by a blank sheath.Ring type C4 is a combination matrix/reservoir-type ring, with LNGloaded in the core and dapivirine loaded in the sheath.

FIG. 14A, FIG. 14B, FIG. 14C, and FIG. 14D depict (FIG. 14A) dailydapivirine release, (FIG. 14B) cumulative dapivirine release, (FIG. 14C)daily LNG release, and (FIG. 15D) cumulative LNG release from ring typeC1, which is a matrix-type ring comprising both DAP and LNG.

FIG. 15A, FIG. 15B, FIG. 15C, and FIG. 15D depict (FIG. 15A) dailydapivirine release, (FIG. 15B) cumulative dapivirine release, (FIG. 15C)daily LNG release, and (FIG. 15D) cumulative LNG release from ring typesC2 (reservoir-type ring with both DAP and LNG loaded in the core,surrounded by a blank sheath), and C3 (reservoir-type ring with DAP andLNG each loaded in separate half-cores, surrounded by a blank sheath).

FIG. 16A and FIG. 16B depict (FIG. 16A) daily DAP and LNG release and(FIG. 16B) cumulative DAP and LNG release, respectively, from ring typeC4 which is a reservoir-matrix-type ring with LNG loaded in the core andDAP loaded in the sheath.

FIG. 17 depicts maraviroc release as a function of time in intravaginalmatrix-type rings comprising 25 mg dapivirine and 100 mg of anantimicrobial compound, maraviroc.

FIG. 18 depicts dapivirine release as a function of time in intravaginalmatrix-type rings comprising 25 mg dapivirine and 100 mg of anantimicrobial compound, maraviroc.

FIG. 19A and FIG. 19B depict the cumulative release of (FIG. 19A)maraviroc and (FIG. 19B) dapivirine from matrix-type intravaginal ringscomprising either 16 mg maraviroc alone, 15 mg dapivirine alone, or 16mg maraviroc and 16 mg dapivirine.

FIG. 20A and FIG. 20B depict the mean daily release of (FIG. 20A)maraviroc and (FIG. 20B) dapivirine from matrix-type intravaginal ringscomprising either 16 mg maraviroc alone, 15 mg dapivirine alone, or 16mg maraviroc and 16 mg dapivirine.

FIG. 21 depicts the phase behavior of maraviroc and dapivirine as afunction of temperature and composition. The liquidus is the maximumtemperature at which crystals can co-exist with the melt inthermodynamic equilibrium. Curves are based on freezing point depressingof each component due to the presence of the other component. Theintersection is the estimated eutectic composition (17.5% dapivirine)and temperature (170° C.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides combination therapy intravaginal drugdelivery devices, i.e., intravaginal rings, useful for theadministration of therapeutic and/or prophylactic agents to a human. Theintravaginal rings of the invention may provide long-term controlledrelease of dapivirine and an antimicrobial agent, such as maraviroc,DS003, darunavir, GSK1265744 or BMS-663068, or dapivirine and acontraceptive, such as levonorgestrel, estradiol, etonorgestrel ornestorone. Surprisingly, the combination of dapivirine and either anantimicrobial compound or a contraceptive in the intravaginal rings ofthe invention results in the formation of a eutectic composition andprovides increased release rates as compared to previous intravaginalrings.

As used herein, the term “intravaginal ring” or “vaginal ring” refers toa doughnut-shaped polymeric drug delivery device which is designed to beinserted into the vagina of a female human in order to providecontrolled release of drugs to the vagina over an extended period oftime. Several single-indication intravaginal rings are currentlyavailable, including Estring® and Femring®, for the treatment ofsymptoms of post-menopause, and NuvaRing®, a contraceptive vaginal ring.

The intravaginal rings of the instant invention provide controlledrelease of dapivirine, alone or in combination with an antimicrobialcompound or a contraceptive, and may have any shape and be of anydimensions compatible with intravaginal administration to a femalehuman. Such a ring can be self-inserted into the vagina, where it isheld in place due to its shape and inherent elasticity. Such a deviceprovides high user adherence, ease of application and exhibits noleakage or messiness on insertion and subsequent placement within thevaginal space.

As used herein, the term “dapivirine” refers to(4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile),a non-nucleoside reverse transcriptase inhibitor. Dapivirine is usefulin the prevention and/or treatment of retroviral infection, such asHIV-1 infection. Dapivirine is a crystalline compound that is white toslightly beige in color, has a melting point of about 220° C. and isvirtually insoluble in water. More specifically, the solubility ofdapivirine is less than 0.001 mg/mLf of water (i.e., less than 1 μg/mlof water). The intravaginal rings of the instant invention may usemicronized dapivirine. A composite result (four samples taken ofmicronized material) showed that 88.15% of the material had a particlesize of less than 5 microns (μM).

As used herein, the term “antimicrobial compound” or “antimicrobialagent” (used interchangeably herein) refers to a compound or agent whichis capable of inhibiting or destroying the growth of a microbialorganism. In a preferred embodiment of the invention, the antimicrobialcompound is a non-nucleoside reverse transcriptase inhibitor (“NNRTI”).In another embodiment, the NNRTI is a substituted di-amino pyrimidinederivative. In another embodiment, the antimicrobial compound is a viralentry inhibitor. In one embodiment of the invention, the antimicrobialcompound is maraviroc. In one embodiment of the invention, theantimicrobial is DS003. In another embodiment of the invention, theantimicrobial compound is darunavir, GSK 1265744 or BMS-663068. The term“antimicrobial compound” is intended to embrace antibacterial agents,antifungal agents, antiprotozoal agents, antiviral agents and mixturesthereof. For purposes of this invention, the term “antimicrobialcompound” or “antimicrobial agent” is a compound other than dapivirine.That is, the intravaginal rings of the invention do not contain onlydapivirine as the active agent.

As used herein, the term “contraceptive” refers to an active agent thatprevents conception or pregnancy. Contraceptives are well-known in theart and include, but are not limited to,17a-ethinyl-levonorgestrel-17b-hydroxy-estra-4,9,11-trien-3-one,estradiol, etonogestrel, levonorgestrel, medroxyprogesterone acetate,nestorone, norethindrone, and progesterone. In one embodiment of theinvention, the contraceptive is levonorgestrel. In one embodiment of theinvention, the contraceptive is estradiol. In one embodiment of theinvention, the contraceptive is etonorgestrel. In one embodiment of theinvention, the contraceptive is nestorone.

As used herein, the term “eutectic composition” refers to a mixture ofchemical compounds that have a single chemical composition thatsolidifies at a lower temperature than any other composition made up ofthe same ingredients. The composition is known as the “eutecticcomposition” and the temperature at which it solidifies is known as the“eutectic temperature.” On a phase diagram, the intersection of theeutectic temperature and the eutectic composition gives the “eutecticpoint.” Non-eutectic mixtures will display solidification of onecomponent of the mixture before the other.

As used herein, the term “matrix ring” or “matrix-type ring” refers toan intravaginal ring in which the active agent or agents arehomogenously distributed throughout the ring. Matrix rings are typicallymanufactured by injection molding or extrusion of a compound-containingactive mix, leading to the uniform distribution of the active compoundthroughout the ring. The matrix-type rings of the instant invention maycomprise dapivirine and either an antimicrobial agent or a contraceptiveagent dispersed in silicone elastomer with normal propylorthosilicate(NPOS) crosslinker. This active mix is subsequently cured using acatalyst, such as platinum (with curing achieved by an additionreaction). The matrix-type rings of the invention may also comprisepolyurethane or EVA. Matrix-type intravaginal rings permit singleintravaginal dosing of active agent(s), with an initially high “loading”and a subsequent, lower “maintenance” release profile.

As used herein, the term “reservoir ring” refers to an intravaginal ringcomprising a reservoir (a full or partial-length core), which iscompletely surrounded by a sheath. In one embodiment, both dapivirineand an antimicrobial compound or a contraceptive compound are present inthe core of a reservoir ring, with a blank sheath. In anotherembodiment, dapivirine and either an antimicrobial compound or acontraceptive are present in separate half-cores (or partial cores) of areservoir ring, with a blank sheath. In yet another embodiment, eitherthe antimicrobial compound or the contraceptive is present in the coreof a reservoir ring, and the dapivirine is present in the sheath.Different types of reservoir ring configurations are depicted in FIG. 13. The release of drug substances from such rings is dependent uponpermeation (i.e., molecular dissolution and subsequent diffusion) of thecore-loaded drug substance through the outer sheath. Release rates canbe modified by changing the nature or thickness of the rate-controllingsheath. Reservoir rings were developed to provide controlled (that is,constant daily) release rates.

As used herein, the term “platinum-catalyzed” refers to an intravaginalring whose cross-linking reaction has been catalyzed using anorgano-platinum compound. In one embodiment, the intravaginal ringcomprises a silicone elastomer. In yet another embodiment, theintravaginal ring comprises a silicone elastomer and a siliconedispersant. The intravaginal ring may comprise other pharmaceuticallycompatible agents. Such agents include pharmacologically active agents,as well as, pharmacologically inactive agents known in the art aspharmaceutical excipients.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Intravaginal Rings

The present invention provides combination therapy intravaginal drugdelivery devices, i.e., intravaginal rings, useful for theadministration of therapeutic and/or prophylactic agents to a human. Theintravaginal rings of the invention may provide long-term controlledrelease of dapivirine and an antimicrobial agent, such as maraviroc,DS003, darunavir, GSK1265744 or BMS-663068, or dapivirine and acontraceptive, such as levonorgestrel, estradiol, etonorgestrel ornestorone. Surprisingly, the combination of dapivirine and either anantimicrobial compound or a contraceptive in the intravaginal rings ofthe invention results in the formation of a eutectic composition andprovides increased release rates as compared to previous intravaginalrings.

As used herein, the term “intravaginal ring” or “vaginal ring” refers toa doughnut-shaped polymeric drug delivery device which is designed to beinserted into the vagina of a female human in order to providecontrolled release of drugs to the vagina over an extended period oftime. Several single-indication intravaginal rings are currentlyavailable, including Estring® and Femring®, for the treatment ofurogenital symptoms of post-menopause, and NuvaRing®, a contraceptivevaginal ring. Intravaginal rings are described in U.S. Pat. No.6,951,654, U.S. Patent Application Publication Nos. US2007/0043332 andUS2009/0004246, PCT Publication Nos. WO99/50250, WO02/076426 andWO03/094920, the entire contents of each of which are expresslyincorporated herein by reference.

The intravaginal rings of the instant invention provide controlledrelease of dapivirine in combination with either an antimicrobialcompound or a contraceptive, and may have any shape and be of anydimensions compatible with intravaginal administration to a femalehuman. Such a ring can be self-inserted into the vagina, where it isheld in place due to its shape and inherent elasticity. In oneembodiment, the intravaginal ring has an outer diameter of 56 mm. Inanother embodiment, the intravaginal ring has an outer diameter of about50 mm, about 51 mm, about 52 mm, about 53 mm, about 54 mm, about 55 mm,about 56 mm, about 57 mm, about 58 mm, about 59 mm or about 60 mm. Inanother embodiment, the intravaginal ring has a cross-sectional diameterof 7.7 mm. In yet another embodiment, the intravaginal ring has across-sectional diameter of about 7.0 mm, about 7.1 mm, about 7.2 mm,about 7.3 mm, about 7.4 mm, about 7.5 mm, about 7.6 mm, about 7.7 mm,about 7.8 mm, about 7.9 mm, about 8.0 mm, about 8.1 mm, about 8.2 mm,about 8.3 mm, about 8.4 mm, or about 8.5 mm.

Such an intravaginal ring permits single intravaginal dosing ofdapivirine and either an antimicrobial agent or a contraceptive, with astable release profile. In addition, a device that can be applied lessfrequently is likely be more acceptable and to achieve better adherencerelative to gels that need to be used more frequently

In one embodiment, the intravaginal ring comprises a silicone elastomer.In yet another embodiment, the intravaginal ring comprises a siliconeelastomer and a silicone dispersant. In another embodiment, theintravaginal ring comprises a polyurethane thermoplastic polymer or anEVA polymer.

The intravaginal ring may comprise other pharmaceutically compatibleagents. Such agents include pharmacologically active agents, as well as,pharmacologically inactive agents known in the art as pharmaceuticalexcipients. Examples of pharmacologically active agents that may beadvantageous include, but are not limited to, a local anesthetic such aslidocaine or a local analgesic or a mixture thereof. Examples ofpharmacologically inactive agents that may be advantageous include, butare not limited to, a buffer (or buffers), or hydrophilic compounds thatenhance the rate of release of the agent from the device, such as forexample, polyvinylpyrrolidone (PVP or povidone), modified celluloseethers (e.g., hydroxyethylcellulose, hydroxypropylcellulose andhydroxypropylmethylcellulose) microcrystalline cellulose, polyacrylicacid, carbomer, alginic acid, carrageenan, cyclodextrins, dextrin, guargum, gelatin, xanthan gum and sugars (e.g., monosaccharides such asglucose, fructose and galactose, and dissaccharides such as lactose,maltose and fructose). When employed, the release rate enhancingexcipient is generally present in an amount of about 0.5 to about 40 w/w% and preferably about 2.5 to about 15 w/w % of the device.

As used herein, the term “matrix ring” or “matrix-type ring” refers toan intravaginal ring in which dapivirine and either an antimicrobialagent or a contraceptive are homogenously distributed throughout thering. Matrix rings are typically manufactured by injection molding orextrusion of the active compound-containing active mix, leading to theuniform distribution of the active compounds throughout the ring. Thematrix-type rings of the instant invention may comprise dapivirine andeither an antimicrobial compound or a contraceptive dispersed insilicone elastomer with normal propylorthosilicate (NPOS) crosslinker.This active mix is subsequently cured using a catalyst, such as platinum(with curing achieved by an addition reaction). Matrix-type rings mayalternatively comprise a polyurethane or EVA polymer.

As used herein, the term “reservoir ring” refers to an intravaginal ringcomprising a reservoir (a full or partial-length core), which iscompletely surrounded by a sheath. In one embodiment, both dapivirineand an antimicrobial compound or a contraceptive compound are present inthe core of a reservoir ring, with a blank sheath. In anotherembodiment, dapivirine and either an antimicrobial compound or acontraceptive are present in separate half-cores of a reservoir ring,with a blank sheath. In yet another embodiment, either the antimicrobialcompound or the contraceptive is present in the core of a reservoirring, and the dapivirine is present in the sheath. Examples of differenttypes of reservoir-type rings are depicted in FIG. 13 . The release ofdrug substances from such rings is dependent upon permeation (i.e.,molecular dissolution and subsequent diffusion) of the core-loaded drugsubstance through the outer sheath. Release rates can be modified bychanging the nature or thickness of the rate-controlling sheath.Reservoir rings were developed to provide controlled (that is, constantdaily) release rates.

As used herein, the term “elastomer” refers to an amorphous, orpredominantly amorphous, polymer network formed when a polymer or amixture of polymers undergo cross-linking. Each polymer is comprised ofmonomeric units, which are linked together to form the network. Themonomeric units can comprise carbon, hydrogen, oxygen, silicon, halogen,or a combination thereof.

In some embodiments, the intravaginal ring comprises a polysiloxane. Asused herein, a “polysiloxane” refers to any of various compoundscontaining alternate silicon and oxygen atoms in either a linear orcyclic arrangement usually with one or two organic groups attached toeach silicon atom. For example, polysiloxanes include substitutedpolysiloxanes, and diorganopolysiloxanes such as diarylpolysiloxanes anddialkylpolysiloxanes; an example of the latter is dimethylpolysiloxane.Such dimethylpolysiloxane polymers can be thermoset to the correspondingelastomer by vulcanization with peroxide curing catalysts, e.g., benzoylperoxide or di-p-chlorobenzoyl peroxide at temperatures of about 200° C.and requiring additional heat after treatment as described in U.S. Pat.Nos. 2,541,137; 2,723,966; 2,863,846; 2,890,188; and 3,022,951, theentire contents of each of which are expressly incorporated herein byreference.

An example of a two-component silicone elastomer, which isplatinum-catalyzed at room temperature or under slightly elevatedtemperature and capable of cross-linking, is MED-4870 (NuSil TechnologyLLC, Carpinteria, Calif.). In some embodiments of the present invention,an intravaginal ring can comprise silicone liquid (NuSil MED360) as adispersing agent, and NuSil MED-4870 elastomer. The MED-4870 elastomeris composed of two parts, part A and part B. The chemical composition ofMED-4870 part A comprises vinyl terminated polydimethylsiloxane (linear)polymers as a polymer, platinum-siloxane complex as the catalyst for thecross-linking reaction, and ˜30% amorphous (non crystalline) reinforcingsilica as a filler. The chemical composition of MED-4870 part Bcomprises vinyl-terminated polydimethylsiloxane (linear) polymers,hydride functional polydimethysiloxane polymer as a cross-linker, and˜30% amorphous (non-crystalline) reinforcing silica as a filler. Form Aand form B undergo cross-linking to form a silicone elastomer.

In some embodiments of the present invention, the polysiloxane elastomeris a diorganopolysiloxane elastomer. In some embodiments, thediorganopolysiloxane elastomer is dimethylpolysiloxane elastomer. Insome embodiments, the dimethylpolysiloxane elastomer further comprises adimethylmethylhydrogen polysiloxane cross-link. In some embodiments ofthe present invention, the polysiloxane elastomer is MED-4870.

In some embodiments, the polysiloxane elastomer is present in aconcentration of about 90% to about 99% by total weight of the ring. Insome embodiments, the polysiloxane elastomer is present in aconcentration of about 95% by total weight of the ring, or about 97% bytotal weight of the ring.

Suitable cross-linking agents and curing catalysts are well known in theart. Curing temperatures and times will vary, depending on theparticular elastomer(s) used. For example, the curing temperature mayvary between room temperature (15-25° C.) and 160° C. but is preferablywithin the range 60-200° C. The curing time may vary between a fewseconds and several hours, depending on the elastomer(s) used. Preferredand suitable elastomers include two-component dimethylpolysiloxanecompositions using platinum as the curing catalyst and at a curingtemperature of from room temperature to an elevated temperature.

As used herein, the term “platinum-catalyzed” refers to an intravaginalring whose cross-linking reaction has been catalyzed using anorgano-platinum compound.

As used herein, the term “alcohol by-product” refers to a volatileby-product of alcohol (including propanol) produced by tin-catalyzedcondensation reactions for cross-linking of solid state silicone.Alcohol by-product contributes to an increased rate of migration ofantimicrobial compound from within the matrix of an intravaginal ring tothe surface, resulting in the undesirable formation of crystallinedeposits of antimicrobial compound on the intravaginal ring.

As used herein, the term “crystalline deposits” refers to theundesirable formation of deposits of crystals of dapivirine, theantimicrobial compound or the contraceptive on the surface of theintravaginal ring.

As used herein, the term “release” or “release rate” refers to theamount or concentration of active agent (i.e., dapivirine, orantimicrobial compound or contraceptive) which leaves the intravaginalring in any defined time period. “Sustained release” or “sustainedrelease rate” refers to release sufficient to provide antimicrobialproperties or contraceptive properties over a specific time period.Release rates of dapivirine, the antimicrobial compound and thecontraceptive are defined in more detail in the subsections, below. Forexample, in one embodiment of the invention, the intravaginal rings aredesigned to provide sustained release of dapivirine and either theantimicrobial compound or the contraceptive. In one embodiment, lessthan about 7 mg of dapivirine is released in vitro during an initial 24hour period of release. In a preferred embodiment of the invention,between about 1 mg and about 3 mg of dapivirine is released in vitroduring an initial 24 hour period of release. In one embodiment, lessthan 1 mg of dapivirine is released in vitro each day for 23 days afteran initial 7 day period of release.

In one embodiment, about 100 μg to about 700 μg of dapivirine isreleased in vitro each day for 23 days after an initial 7 day period ofrelease. In another embodiment, about 100 μg, about 125 μg, about 150μg, about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275μg, about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500μg, about 550 μg, about 600 μg, about 650 μg, or about 700 μg ofdapivirine is released in vitro each day for 23 days after an initial 7day period of release.

In another embodiment, less than 1 mg of dapivirine is released in vitroeach day for 53 days after an initial 7 day period of release. In oneembodiment, about 100 μg to about 700 μg of dapivirine is released invitro each day for 53 days after an initial 7 day period of release. Inanother embodiment, about 100 μg, about 125 μg, about 150 μg, about 175μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, about 550μg, about 600 μg, about 650 μg, or about 700 μg of dapivirine isreleased in vitro each day for 53 days after an initial 7 day period ofrelease.

In another embodiment, between about 5 μg and about 300 μg of dapivirineis released in vivo per gram of vaginal fluid each day for 24 days afteran initial 3 day period of use. In another embodiment, between about 10μg and about 100 μg of dapivirine is released in vivo per gram ofvaginal fluid each day for 24 days after an initial 3 day period of use.In another embodiment, between about 20 μg and about 80 μg of dapivirineis released in vivo per gram of vaginal fluid each day for 24 days afteran initial 3 day period of use. In yet another embodiment, about 5 μg,about 10 μg, about 25 μg, about 50 μg, about 75 μg, about 100 μg, about125 μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about250 μg, about 275 μg, about 300 μg, about 350 μg, about 400 μg, about450 μg, about 500 μg, or about 600 μg of dapivirine s released in vivoper gram of vaginal fluid each day for 24 days after an initial 3 dayperiod of use.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure. Moreover, the amount of dapivirine, antimicrobial compound,or contraceptive released may clearly be varied depending on, forexample, the desired dosing level, the particular compound, the releaserate effect of excipients used in the device, and the particularelastomeric system employed.

As used herein, the term “initial 24 hour period of use” refers to thefirst day, or twenty-four hours, of time after the initial use of theintravaginal ring. The initial 24 hour period of use begins when theintravaginal ring is inserted into the vagina of the female human.

As used herein, the term “each day” refers to an individual 24 hourperiod.

In one embodiment, less than about 2 mg of the antimicrobial compound isreleased from the ring in vitro during an initial 24 hour period ofrelease. In another embodiment, less than about 1 mg of theantimicrobial compound is released from the ring in vitro during aninitial 24 hour period of release.

In one embodiment, the antimicrobial compound is released in vitro at arate of about 200 μg per day to about 8000 μg per day for about 23 daysafter an initial 7 day period of release. In another embodiment, theantimicrobial compound is released in vitro at a rate of about 200 μgper day to about 8000 μg per day for about 53 days after the initial 7day period of release. In another embodiment, the antimicrobial compoundis released in vitro at a rate of about 200 μg per day to about 2000 μgper day, about 400 μg per day to about 4000 μg per day, about 550 μg perday to about 5500 μg per day, or about 800 μg per day to about 8000 μgper day for about 23 days or about 53 days after the initial 7 dayperiod of release.

In one embodiment, less than about 8000 μg per day of the antimicrobialcompound is released in vitro after the initial 7 day period of releasefor about 23 days or for about 53 days. In another embodiment, less thanabout 5500 μg per day, 4000 μg per day, or 4000 μg per day of theantimicrobial compound is released in vitro after the initial 7 dayperiod of release for about 23 days or for about 53 days. In oneembodiment, at least about 200 μg per day, 400 μg per day, 550 μg perday, or 800 μg per day of the antimicrobial compound is released invitro after the initial 7 day period of release for about 23 days orabout 53 days.

In one embodiment, the antimicrobial compound is released in vitro at arate of about 200 μg per day to about 2000 μg per day for about 30 daysor for about 60 days. In another embodiment, the antimicrobial compoundis released in vitro at a rate of about 400 μg per day to about 4000 μgper day for about 30 days or for about 60 days. In one embodiment, theantimicrobial compound is released in vitro at a rate of about 550 μgper day to about 5500 μg per day for about 30 days or for about 60 days.In another embodiment, the antimicrobial compound is released in vitroat a rate of about 800 μg per day to about 800 μg per day for about 30days or for about 60 days.

In one embodiment, less than about 2 mg of the contraceptive is releasedfrom the ring in vitro during an initial 24 hour period of release. Inanother embodiment, less than about 1 mg of the contraceptive isreleased from the ring in vitro during an initial 24 hour period ofrelease. In one embodiment, the contraceptive is released in vitro at arate of about 20 μg per day to about 290 μg per day for about 23 daysafter an initial 7 day period of release. In another embodiment, thecontraceptive is released in vitro at a rate of about 20 μg per day toabout 290 μg per day for about 53 days after the initial 7 day period ofrelease. In another embodiment, the contraceptive is released in vitroat a rate of about 35 μg per day to about 70 μg per day for about 23days after the initial 7 day period of release. In another embodiment,the contraceptive is released in vitro at a rate of about 35 μg per dayto about 70 μg per day for about 53 days after the initial 7 day periodof release.

In one embodiment, less than about 100 μg per day of the contraceptiveis released in vitro after the initial 7 day period of release for about23 days. In another embodiment, less than about 100 μg per day of thecontraceptive is released in vitro after the initial 7 day period ofrelease for about 53 days. In one embodiment, less than about 70 μg perday of the contraceptive is released in vitro after the initial 7 dayperiod of release for about 23 days. In one embodiment, less than about70 μg per day of the contraceptive is released in vitro after theinitial 7 day period of release for about 53 days. In one embodiment,less than about 15 μg per day of the contraceptive is released in vitroafter the initial 7 day period of release for about 23 days. In oneembodiment, less than about 15 μg per day of the contraceptive isreleased in vitro after the initial 7 day period of release for about 53days. In one embodiment, about 35 μg per day of the contraceptive isreleased in vitro after the initial 7 day period of release for about 23days or for about 53 days.

In one embodiment, the contraceptive is released in vitro at a rate ofabout 20 μg per day to about 290 μg per day for about 30 days. Inanother embodiment, the contraceptive is released in vitro at a rate ofabout 20 μg per day to about 290 μg per day for about 60 days. In oneembodiment, the contraceptive is released in vitro at a rate of about 35μg per day to about 70 μg per day for about 30 days. In anotherembodiment, the contraceptive is released in vitro at a rate of about 35μg per day to about 70 μg per day for about 60 days.

In one embodiment, the contraceptive is released in vitro at a rate ofless than about 100 μg per day for about 30 days. In another embodiment,the contraceptive is released in vitro at a rate of less than about 100μg per day for about 60 days. In one embodiment, the contraceptive isreleased in vitro at a rate of less than about 70 μg per day for about30 days. In another embodiment, the contraceptive is released in vitroat a rate of less than about 70 μg per day for about 60 days. In oneembodiment, the contraceptive is released in vitro at a rate of lessthan about 35 μg per day for about 30 days. In another embodiment, thecontraceptive is released in vitro at a rate of less than about 35 μgper day for about 60 days. In one embodiment, the contraceptive isreleased in vitro at a rate of less than about 15 μg per day for about30 days. In another embodiment, the contraceptive is released in vitroat a rate of less than about 15 μg per day for about 60 days.

As used herein, the term “homogenously dispersed throughout” refers to acompound which is uniformly distributed throughout the intravaginalring.

As used herein, the term “prophylactically effective amount” refers tothe amount of antimicrobial compound effective to prevent development ofdisease in the subject. In one embodiment of the invention, the diseaseis HIV. In a preferred embodiment of the invention, a prophylacticallyeffective amount is achieved when between about 1 mg and about 3 mg ofdapivirine and/or antimicrobial compound is released in vitro during aninitial 24 hour period of release. In one embodiment of the invention, aprophylactically effective amount is achieved when between about 100 μgto about 700 μg of dapivirine and/or antimicrobial compound is releasedin vitro per day after the initial 7 day period of release. In anotherembodiment, a prophylactically effective amount is achieved when betweenabout 100 μg to about 500 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In one embodiment of the invention, a prophylactically effective amountis achieved when between about 200 μg to about 400 μg of dapivirineand/or antimicrobial compound is released in vitro per day after theinitial 7 day period of release. In another embodiment of the invention,a prophylactically effective amount is achieved when between about 250μg to about 350 μg of dapivirine and/or antimicrobial compound isreleased in vitro per day after the initial 7 day period of release. Inanother embodiment of the invention, a prophylactically effective amountis achieved when between about 300 μg to about 400 μg of dapivirineand/or antimicrobial compound is released in vitro per day after theinitial 7 day period of release. In one embodiment, a prophylacticallyeffective amount is achieved when about 50 μg, about 100 μg, about 125μg, about 150 μg, about 175 μg, about 200 μg, about 225 μg, about 250μg, about 275 μg, about 300 μg, about 325 μg, about 350 μg, about 375μg, about 400 μg, about 450 μg, about 475 μg, about 500 μg, about 550μg, about 600 μg, about 650 μg, about 700 μg, about 750 μg, about 800μg, about 850 μg, about 900 μg about 950 μg, about 1 mg, less than about1 mg or more than about 1 mg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.

In another embodiment, a prophylactically effective amount is achievedwhen between about 5 μg and about 300 μg of dapivirine and/orantimicrobial compound is released in vivo per gram of vaginal fluideach day for 24 days after an initial 3 day period of use. In anotherembodiment, a prophylactically effective amount is achieved when betweenabout 10 μg and about 100 μg of dapivirine and/or antimicrobial compoundis released in vivo per gram of vaginal fluid each day for 24 days afteran initial 3 day period of use. In another embodiment, aprophylactically effective amount is achieved when between about 20 μgand about 80 μg of dapivirine and/or antimicrobial compound is releasedin vivo per gram of vaginal fluid each day for 24 days after an initial3 day period of use. In yet another embodiment, a prophylacticallyeffective amount is achieved when about 5 μg, about 10 μg, about 25 μg,about 50 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg,about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg,about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, orabout 600 μg of dapivirine and/or antimicrobial compound is released invivo per gram of vaginal fluid each day for 24 days after an initial 3day period of use.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

As used herein, the term “therapeutically effective amount” refers tothe amount of compound effective to treat disease in the subject. In oneembodiment of the invention, the disease is HIV. In a preferredembodiment of the invention, a therapeutically effective amount isachieved when less than about 7 mg of dapivirine and/or antimicrobialcompound is released in vitro during an initial 24 hour period of use.In one embodiment, a therapeutically effective amount is achieved whenbetween about 1 mg and about 3 mg of dapivirine and/or antimicrobialcompound is released in vitro during an initial 24 hour period ofrelease. In one embodiment of the invention, a therapeutically effectiveamount is achieved when between about 100 μg to about 700 μg ofdapivirine and/or antimicrobial compound is released in vitro per dayafter the initial 7 day period of release. In one embodiment of theinvention, a therapeutically effective amount is achieved when betweenabout 200 μg to about 400 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In another embodiment of the invention, a therapeutically effectiveamount is achieved when between about 250 μg to about 350 μg ofdapivirine and/or antimicrobial compound is released in vitro per dayafter the initial 7 day period of release. In another embodiment of theinvention, a therapeutically effective amount is achieved when betweenabout 180 μg to about 600 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In one embodiment, a therapeutically effective amount is achieved whenabout 100 μg, about 125 μg, about 150 μg, about 175 μg, about 180 μg,about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg,about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 450 μg,about 475 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg, orabout 700 μg of dapivirine and/or antimicrobial compound is released invitro per day after the initial 7 day period of release.

In another embodiment, a therapeutically effective amount is achievedwhen between about 5 μg and about 300 μg of dapivirine and/orantimicrobial compound is released in vivo per gram of vaginal fluideach day for 24 days after an initial 3 day period of use. In anotherembodiment, a therapeutically effective amount is achieved when betweenabout 10 μg and about 100 μg of dapivirine and/or antimicrobial compoundis released in vivo per gram of vaginal fluid each day for 24 days afteran initial 3 day period of use. In another embodiment, a therapeuticallyeffective amount is achieved when between about 20 μg and about 80 μg ofdapivirine and/or antimicrobial compound is released in vivo per gram ofvaginal fluid each day for 24 days after an initial 3 day period of use.In yet another embodiment, a therapeutically effective amount isachieved when about 5 μg, about 10 μg, about 25 μg, about 50 μg, about75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 350μg, about 400 μg, about 450 μg, about 500 μg, or about 600 μg ofdapivirine and/or antimicrobial compound is released in vivo per gram ofvaginal fluid each day for 24 days after an initial 3 day period of use.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

As used herein, the term “controlled release rate” refers to a constantrelease rate that can be determined by the design and drug loading ofthe vaginal ring.

As used herein, the term “constant release rate” refers to a releaserate which does not readily change with device storage over time.Preferably, the release rate of dapivirine, the antimicrobial compoundor the contraceptive from the intravaginal ring is constant, or stableand does not readily change over time at room temperature (about 30° C.)or at 40° C. for at least 1 month, at about 2-8° C. for at least 1 year,or for at least 2 years. For example, the release rate of dapivirine,the antimicrobial compound or the contraceptive from the intravaginalrings of the instant invention can be stable for 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36,42 or 48 months.

As used herein, the term “steady release rate” means a release rate thatshows relatively little change over time.

A “stable” compound is one which essentially retains its physicalstability and/or chemical stability and/or biological activity duringthe manufacturing process and/or upon storage. Various analyticaltechniques for measuring stability are available in the art and arereviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed.,Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv.Drug Delivery Rev. 10: 29-90 (1993).

As used herein, the term “storage” refers to the period of time afterwhich the intravaginal rings are made, but before which the intravaginalrings are used. For example, the intravaginal rings of the instantinvention can be stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 30, 36, 42 or 48 months.

II. Dapivirine

The instant invention is based on the surprising discovery thatintravaginal rings comprising dapivirine and either an antimicrobialcompound or a contraceptive result in a synergetic composition withenhanced release rates of either the antimicrobial compound, thecontraceptive, or dapivirine, as compared to release rates of thecompounds, alone. As used herein, the term “dapivirine” refers to(4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino]benzonitrile),a non-nucleoside reverse transcriptase inhibitor (see structure, below).

Dapivirine is useful in the prevention and/or treatment of retroviralinfection, such as HIV-1 infection. It is a crystalline compound that iswhite to slightly beige in color, has a melting point of about 220° C.and is virtually insoluble in water. More specifically, the solubilityof dapivirine is less than 0.001 mg/gm of water (i.e., less than 1 μg/mlof water). The intravaginal rings of the instant invention may usemicronized dapivirine. A composite result (four samples taken ofmicronized material) showed that 88.15% of the material had a particlesize of less than 5 microns (μM).

Dapivirine was originally developed as an oral antiretroviral compoundand was first conceived as an oral therapeutic. Dapivirine has potentactivity against wild-type HIV-1 strains and HIV-1 strains harboringdifferent resistance-inducing mutations. (Das et al., J. Med Chem.,2004; 47(10):2550-60.) Dapivirine is a white to off-white or slightlyyellow powder, free from visible impurities, has a melting point ofapproximately 220° C., and is virtually insoluble in water. Dapivirine,a substituted DAPY derivate with the chemical name4-[[4-[(2,4,6-trimethylphenyl)amino]-2-pyrimidinyl]amino] benzonitrile,is a non-nucleoside reverse transcriptase inhibitor (NNRTI).

In one embodiment, about 10 to about 30 mg of dapivirine is present inthe ring. In another embodiment, about 20 mg to about 30 mg ofdapivirine is present in the ring. In yet another embodiment, about 10to about 800 mg, about 50 mg to about 750 mg, about 100 mg to about 700mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about 10mg to about 100 mg, about 100 mg to about 200 mg, about 150 mg to about250 mg, about 200 mg to about 600 mg, or about 300 mg to about 400 mg ofdapivirine is present in the ring.

In another embodiment, about 15 mg of dapivirine is present in the ring.In another embodiment, about 25 mg of dapivirine is present in the ring.In another embodiment, about 100 mg of dapivirine is present in thering. In another embodiment, about 150 mg of dapivirine is present inthe ring. In another embodiment, about 250 mg of dapivirine is presentin the ring. In another embodiment, about 5 mg, about 6 mg, about 7 mg,about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13mg, about 14 mg, about 15 mg, about 16 mg, about 17 mg, about 18 mg,about 19 mg, about 20 mg, about 21 mg, about 22 mg, about 23 mg, about24 mg, about 25 mg, about 26 mg, about 27 mg, about 28 mg, about 29 mg,about 30 mg, about 31 mg, about 32 mg, about 33 mg, about 34 mg, about35 mg, about 36 mg, about 37 mg, about 38 mg, about 39 mg, about 40 mg,about 41 mg, about 42 mg, about 43 mg, about 44 mg, about 45 mg, about46 mg, about 47 mg, about 48 mg, about 49 mg, about 50 mg, about 55 mg,about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about90 mg, about 100 mg, about 125 mg, about 150 mg, about 175 mg, about 200mg, about 225 mg, about 250 mg, about 275 mg, about 300 mg, about 350mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600mg, about 650 mg, about 700 mg, about 750 mg, or about 800 mg ofdapivirine is present in the ring.

In one embodiment, the invention provides intravaginal rings comprisingdapivirine and an antimicrobial compound. In another embodiment, theinvention provides intravaginal rings comprising dapivirine and acontraceptive. In another embodiment, the invention providesintravaginal rings comprising dapivirine, an antimicrobial compound, anda contraceptive.

The intravaginal rings of the invention may provide long-term controlledrelease of dapivirine and an antimicrobial agent, such as maraviroc,DS003, darunavir, GSK1265744 or BMS-663068, or dapivirine and acontraceptive, such as levonorgestrel, estradiol, etonorgestrel ornestorone. Surprisingly, the combination of dapivirine and either anantimicrobial compound or a contraceptive in the intravaginal rings ofthe invention results in the formation of a eutectic composition andprovides increased release rates as compared to previous intravaginalrings.

As used herein, the term “eutectic composition” refers to a mixture ofchemical compounds that have a single chemical composition thatsolidifies at a lower temperature than any other composition made up ofthe same ingredients. The composition is known as the “eutecticcomposition” and the temperature at which it solidifies is known as the“eutectic temperature.” On a phase diagram, the intersection of theeutectic temperature and the eutectic composition gives the “eutecticpoint.” Non-eutectic mixtures will display solidification of onecomponent of the mixture before the other.

In one embodiment of the invention, the combination of dapivirine and anantimicrobial compound, e.g., maraviroc, results in the formation of aeutectic composition and provides increased release rate of theantimicrobial compound as compared to intravaginal rings comprising theantimicrobial compound without dapivirine. In one embodiment of theinvention, the combination of dapivirine and an antimicrobial compound,e.g., maraviroc, results in the formation of a eutectic composition andprovides increased release rate of dapivirine as compared tointravaginal rings comprising the antimicrobial compound withoutdapivirine.

In another embodiment of the invention, the combination of dapivirineand a contraceptive, e.g., levonorgestrel, results in the formation of aeutectic composition and provides increased release rate of thecontraceptive as compared to intravaginal rings comprising thecontraceptive without dapivirine. In one embodiment of the invention,the combination of dapivirine and a contraceptive, e.g., levonorgestrel,results in the formation of a eutectic composition and providesincreased release rate of dapivirine as compared to intravaginal ringscomprising the contraceptive without dapivirine.

As used herein, the term “release” or “release rate” refers to theamount or concentration of active agent (i.e., dapivirine, orantimicrobial compound or contraceptive) which leaves the intravaginalring in any defined time period. “Sustained release” or “sustainedrelease rate” refers to release of the active agent sufficient toprovide antimicrobial properties or contraceptive properties over aspecific time period. For example, in one embodiment of the invention,the intravaginal rings are designed to provide sustained release ofdapivirine and either the antimicrobial compound or the contraceptive.In a preferred embodiment of the invention, less than about 7 mg ofdapivirine is released in vitro during an initial 24 hour period ofrelease. In another embodiment, between about 1 mg and about 3 mg ofdapivirine is released in vitro during an initial 24 hour period ofrelease. In one embodiment, less than 1 mg of dapivirine is released invitro each day for 23 days after an initial 7 day period of release.

As used herein, the term “increased release” or “increased release rate”refers to the release or the release rate of an active agent (e.g.,dapivirine, an antimicrobial compound, or a contraceptive) from anintravaginal ring of the invention comprising a combination ofdapivirine and either an antimicrobial compound or a contraceptive thatis greater, or larger, than the release or the release rate of theactive agent (e.g., dapivirine, an antimicrobial compound, or acontraceptive) from an intravaginal ring comprising only one activeagent, e.g., dapivirine only, an antimicrobial agent only, or acontraceptive only. Increased release rates can be expressed in terms ofincreased release per day or cumulative release rate over a period oftime, e.g., increased cumulative release over 30 days, over 60 days, orover 90 days. Increased release rates can be expressed in terms ofincreased release per day after a period of time, e.g., increasedcumulative release over 23 days, 53 days or 83 days after an initial 7day period of release. Increased release rates can also be expressed interms of increased release per day after a period of time, e.g.,increased cumulative release over 29 days, 59 days or 89 days after aninitial 24 hour period of release.

In one of the invention, the release rate of the dapivirine from acombination therapy intravaginal ring of the invention (e.g., a ringcomprising dapivirine and either an antimicrobial agent, e.g.,maraviroc, or a contraceptive, e.g., levonorgestrel) is not increased ascompared to the release rate of dapivirine from an intravaginal ringcomprising only dapivirine. In one embodiment, the release rate of theantimicrobial compound or the contraceptive is increased from thecombination therapy intravaginal ring of the invention as compared tothe release rate of the antimicrobial compound or the contraceptive froman intravaginal ring comprising dapivirine without either theantimicrobial compound or the contraceptive.

In another embodiment, the release rate of the dapivirine from acombination therapy intravaginal ring of the invention (e.g., a ringcomprising dapivirine and either an antimicrobial agent, e.g.,maraviroc, or a contraceptive, e.g., levonorgestrel) is increased ascompared to the release rate of dapivirine from an intravaginal ringcomprising dapivirine without either the antimicrobial compound or thecontraceptive. For example, in one embodiment, the release rate ofdapivirine from an intravaginal ring of the invention comprisingdapivirine and either an antimicrobial agent or a contraceptive isincreased at least about 2-fold per day as compared to the release rateof dapivirine from an intravaginal ring comprising dapivirine withouteither the antimicrobial agent or the contraceptive. In one embodiment,the release rate of dapivirine from an intravaginal ring of theinvention comprising dapivirine and either an antimicrobial agent or acontraceptive is increased at least about 10-fold per day as compared tothe release rate of dapivirine from an intravaginal ring comprisingdapivirine without either the antimicrobial agent or the contraceptive.In another embodiment, the release rate of dapivirine from anintravaginal ring of the invention comprising dapivirine and either anantimicrobial agent or a contraceptive is increased at least about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50-fold per day as compared tothe release rate of dapivirine from an intravaginal ring comprisingdapivirine without either the antimicrobial agent or the contraceptive.In one embodiment, the release rate of dapivirine from an intravaginalring of the invention comprising dapivirine and either an antimicrobialagent or a contraceptive is increased by about 2-fold to about 50-fold,by about 2-fold to about 25-fold, by about 2-fold to about 20-fold, byabout 2-fold to about 15-fold, by about 2-fold to about 10-fold, byabout 2-fold to about 5-fold, by about 5-fold to about 25-fold, by about5-fold to about 20-fold, by about 5-fold to about 15-fold, by about5-fold to about 10-fold, by about 10-fold to about 25-fold, or by about1-fold to about 20-fold per day as compared to the release rate ofdapivirine from an intravaginal ring comprising dapivirine withouteither the antimicrobial agent or the contraceptive.

In one embodiment, the cumulative release rate of dapivirine from anintravaginal ring of the invention comprising dapivirine and either anantimicrobial agent or a contraceptive agent is increased at least about2-fold after 30 days, after 60 days, or after 90 days as compared to thecumulative release rate of dapivirine from an intravaginal ringcomprising dapivirine without either the antimicrobial agent or thecontraceptive after 30 days, after 60 days, or after 90 days. In oneembodiment, the cumulative release rate of dapivirine from anintravaginal ring of the invention comprising dapivirine and either anantimicrobial agent or a contraceptive is increased at least about10-fold after 30 days, after 60 days, or after 90 days as compared tothe cumulative release rate of dapivirine from an intravaginal ringcomprising dapivirine without either the antimicrobial agent or thecontraceptive after 30 days, after 60 days, or after 90 days. In anotherembodiment, the cumulative release rate of dapivirine from anintravaginal ring of the invention comprising dapivirine and either anantimicrobial agent or a contraceptive is increased at least about 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50-fold after 30 days, after60 days, or after 90 days as compared to the cumulative release rate ofdapivirine from an intravaginal ring comprising dapivirine without theantimicrobial agent or the contraceptive after 30 days, after 60 days,or after 90 days. In one embodiment, the cumulative release rate ofdapivirine from an intravaginal ring of the invention comprisingdapivirine and either an antimicrobial agent or a contraceptive isincreased by about 2-fold to about 50-fold, by about 2-fold to about25-fold, by about 2-fold to about 20-fold, by about 2-fold to about15-fold, by about 2-fold to about 10-fold, by about 2-fold to about5-fold, by about 5-fold to about 25-fold, by about 5-fold to about20-fold, by about 5-fold to about 15-fold, by about 5-fold to about10-fold, by about 10-fold to about 25-fold, or by about 1-fold to about20-fold after 30 days, after 60 days, or after 90 days as compared tothe cumulative release rate of dapivirine from an intravaginal ringcomprising dapivirine without the antimicrobial agent or thecontraceptive after 30 days, after 60 days, or after 90 days.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

III. Antimicrobial Compounds

The instant invention is based on the surprising discovery thatintravaginal rings comprising dapivirine and an antimicrobial compoundresult in a synergetic composition with enhanced release rates of eitherthe antimicrobial compound or dapivirine, as compared to release ratesof the compounds, alone.

As used herein, the term “antimicrobial compound” or “antimicrobialagent” (used interchangeably herein) refers to a compound or agent whichis capable of inhibiting or destroying the growth of a microbialorganism. In one embodiment, the antimicrobial compound is anon-nucleoside reverse transcriptase inhibitor (“NNRTI”). In oneembodiment, the antimicrobial compound is a viral entry inhibitor. Inone embodiment of the invention, the antimicrobial compound ismaraviroc. In one embodiment of the invention, the antimicrobial isDS003. In another embodiment of the invention, the antimicrobialcompound is darunavir, GSK 1265744 or BMS-663068. The term“antimicrobial compound” is intended to embrace antibacterial agents,antifungal agents, antiprotozoal agents, antiviral agents and mixturesthereof. For purposes of this invention, the term “antimicrobialcompound” or “antimicrobial agent” is a compound other than dapivirine.That is, the intravaginal rings of the invention do not contain onlydapivirine as the active agent.

In another embodiment of the invention, the antimicrobial compound is anon-nucleoside reverse transcriptase inhibitor (“NNRTI”). In oneembodiment, the NNRTI is a substituted di-amino pyrimidine derivative.Useful NNRTI class compounds include, but are not limited to,nevirapine, delavirdine, etravirine and efavirenz. NNRTIs bind to thehydrophobic pocket near the active site of the HIV reverse transcriptase(RT) enzyme, blocking DNA polymerization. (See, e.g., Tarby, Curr. Top.Med. Chem., 2004; 4(10):1045-57, U.S. Patent Application Publication No.US2006/0166943, and PCT Publication No. WO03/094920, the entire contentsof each of which are expressly incorporated herein by reference.) Thisprevents viral replication and, therefore, production of infectiousvirus. (Borkow et al., J. Virol., 1997; 71(4):3023-30.)

In another preferred embodiment of the invention, the antimicrobialcompound is a viral entry inhibitor. In another embodiment, the viralentry inhibitor is maraviroc. In another preferred embodiment of theinvention, a nucleoside reverse transcriptase inhibitor is used.

The term “antimicrobial compound” is intended to embrace antibacterialagents, antifungal agents, antiprotozoal agents, antiviral agents andmixtures thereof.

Suitable antibacterial agents include Acrosoxacin, Amifloxacin,Amoxycillin, Ampicillin, Aspoxicillin, Azidocillin, Azithromycin,Aztreonam, Balofloxacin, Benzylpenicillin, Biapenem, Brodimoprim,Cefaclor, Cefadroxil, Cefatrizine, Cefcapene, Cefdinir, Cefetamet,Cefmetazole, Cefprozil, Cefroxadine, Ceftibuten, Cefuroxime, Cephalexin,Cephalonium, Cephaloridine, Cephamandole, Cephazolin, Cephradine,Chlorquinaldol, Chlortetracycline, Ciclacillin, Cinoxacin,Ciprofloxacin, Clarithromycin, Clavulanic Acid, Clindamycin,Clofazimine, Cloxacillin, Danofloxacin, Dapsone, Demeclocycline,Dicloxacillin, Difloxacin, Doxycycline, Enoxacin, Enrofloxacin,Erythromycin, Fleroxacin, Flomoxef, Flucloxacillin, Flumequine,Fosfomycin, Isoniazid, Levofloxacin, Mandelic Acid, Mecillinam,Metronidazole, Minocycline, Mupirocin, Nadifloxacin, Nalidixic Acid,Nifuirtoinol, Nitrofurantoin, Nitroxoline, Norfloxacin, Ofloxacin,Oxytetracycline, Panipenem, Pefloxacin, Phenoxymethylpenicillin,Pipemidic Acid, Piromidic Acid, Pivampicillin, Pivmecillinam,Prulifloxacin, Rufloxacin, Sparfloxacin, Sulbactam, Sulfabenzamide,Sulfacytine, Sulfametopyrazine, Sulphacetamide, Sulphadiazine,Sulphadimidine, Sulphamethizole, Sulphamethoxazole, Sulphanilamide,Sulphasomidine, Sulphathiazole, Temafloxacin, Tetracycline, Tetroxoprim,Tinidazole, Tosufloxacin, Trimethoprim and salts or esters thereof.

Preferred antibacterial agents include tetracyclines such asDoxycycline, Tetracycline or Minocycline; macrolides such asAzithromycin, Clarithromycin and Erythromycin; nitroimidazoles such asMetronidazole or Tinidazole; quinolones such as Ofloxacin, Norfloxacin,Cinoxacin, Ciprofloxacin and Levofloxacin; Clindamycin and Dapsone.

Suitable antifungal agents include Bifonazole, Butoconazole,Chlordantoin, Chlorphenesin, Ciclopirox Olamine, Clotrimazole,Eberconazole, Econazole, Fluconazole, Flutrimazole, Isoconazole,Itraconazole, Ketoconazole, Miconazole, Nifuroxime, Tioconazole,Terconazole, Undecenoic Acid and salts or esters thereof.

Preferred antifungal agents include Clotrimazole, Econazole,Fluconazole, Itraconazole, Ketoconazole, Miconazole, Terconazole andTioconazole.

Suitable antiprotozoal agents include Acetarsol, Azanidazole,Chloroquine, Metronidazole, Nifuratel, Nimorazole, Omidazole,Propenidazole, Secnidazole, Sineflngin, Tenonitrozole, Temidazole,Tinidazole and salts or esters thereof.

Metronidazole, Tinidazole and Chloroquine are most preferredantiprotozoal agents.

Suitable antiviral agents include Acyclovir, Brivudine, Cidofovir,Curcumin, Dapirivine, Desciclovir, 1-Docosanol, Edoxudine, Fameyclovir,Fiacitabine, Ibacitabine, Imiquimod, Lamivudine, Penciclovir,Valacyclovir, Valganciclovir and salts or esters thereof. Curcumin,Acyclovir, Famcyclovir, Dapirivine and Valacyclovir are preferredantiviral agents.

The most preferred antimicrobial agents of this invention include,without limitation, Dapirivine, Metronidazole, Acyclovir, Clotrimazole,Fluconazole, Terconazole, Azithromycin, Erythromycin, Doxycycline,Tetracycline, Minocycline, Clindamycin, Famcyclovir, Valacyclovir,Clarithromycin, a prodrug or salt thereof and combinations thereof.

Mixtures of antibacterial agents, mixtures of antifungal agents;mixtures of antiviral agents; mixtures of antiprotozoal agents andmixtures of agents from two or more of these categories are alsoenvisaged by the present invention. In addition, it is also envisagedthat the present invention embraces at least one antimicrobial agent(microstatic and/or microcidal agent) with one or more otherpharmaceutically active agent.

In one embodiment of the invention, the intravaginal ring comprisesdapivirine and one antimicrobial agent. In another embodiment of theinvention, the intravaginal ring comprises dapivirine and at least two,at least three, at least four, or at least five antimicrobial agents. Inanother embodiment of the invention, the intravaginal ring comprisesdapivirine, a antimicrobial agent, and a contraceptive.

Antimicrobial compounds contained in the rings of the present inventionare further described at least in U.S. Patent Application PublicationNos. 2012/0093911 and 2006/0166943 and PCT Publication Nos. WO99/50250,WO02/076426 and WO03/094920, the entire contents of each of which areexpressly incorporated herein by reference. The antimicrobial compoundscontained in the rings of the present invention can be preparedaccording to art-known procedures. In particular, they are preparedaccording to the procedures described in EP 1002795, WO 99/50250, WO99/50256 and WO 00/27828, the entire contents of each of which areincorporated herein by reference.

The antimicrobial compounds contained in the rings of the presentinvention may have microbicidal activity and have the ability to preventthe transmission of HIV. In particular, they can prevent sexual orvaginal transmission of HIV by preventing either the production ofinfectious viral particles or infection of uninfected cells. If infectedcells in sperm can reach the mucosa, the compounds of the presentinvention can prevent HIV infection of host cells, such as macrophages,lymphocytes, Langerhans and M cells. Thus, these compounds preventsystemic HIV infection of a human being, exhibiting a prophylacticaction against HIV.

In one embodiment, about 10 to about 30 mg of antimicrobial agent ispresent in the ring. In another embodiment, about 20 mg to about 30 mgof antimicrobial agent is present in the ring. In yet anotherembodiment, about 10 to about 800 mg, about 50 mg to about 750 mg, about100 mg to about 700 mg, or about 200 mg to about 600 mg, about 300 mg toabout 400 mg, or about 100 mg to about 1600 mg of antimicrobial agent ispresent in the ring.

In another embodiment, about 15 mg, 16 mg, 25 mg, 32 mg, 100 mg, 150 mgor 250 mg of antimicrobial agent is present in the ring. In anotherembodiment, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg,about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about15 mg, about 16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg,about 21 mg, about 22 mg, about 23 mg, about 24 mg, about 25 mg, about26 mg, about 27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg,about 32 mg, about 33 mg, about 34 mg, about 35 mg, about 36 mg, about37 mg, about 38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg,about 43 mg, about 44 mg, about 45 mg, about 46 mg, about 47 mg, about48 mg, about 49 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg,about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 100 mg, about125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about250 mg, about 275 mg, about 300 mg, about 350 mg, about 400 mg, about450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about700 mg, about 750 mg, about 800 mg, about 900 mg, about 1000 mg, about1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, orabout 1600 mg of antimicrobial agent is present in the ring.

Surprisingly, the combination of dapivirine and an antimicrobialcompound, e.g., maraviroc, in the intravaginal rings of the inventionresults in the formation of a eutectic composition and provides anincreased release rate of the antimicrobial compound as compared to therelease rate of the antimicrobial compound in intravaginal ringscomprising the antimicrobial compound without dapivirine. For example,in one embodiment, the release rate of the antimicrobial agent from anintravaginal ring of the invention comprising dapivirine and theantimicrobial agent is increased at least about 2-fold per day ascompared to the release rate of the antimicrobial agent from anintravaginal ring comprising the antimicrobial agent without dapivirine.In one embodiment, the release rate of the antimicrobial agent from anintravaginal ring of the invention comprising dapivirine and theantimicrobial agent is increased at least about 10-fold per day ascompared to the release rate of the antimicrobial agent from anintravaginal ring comprising the antimicrobial agent without dapivirine.In another embodiment, the release rate of the antimicrobial agent froman intravaginal ring of the invention comprising dapivirine and theantimicrobial agent is increased at least about 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45 or 50-fold per day as compared to the release rate ofthe antimicrobial agent from an intravaginal ring comprising theantimicrobial agent without dapivirine. In one embodiment, the releaserate of the antimicrobial agent from an intravaginal ring of theinvention comprising dapivirine and the antimicrobial agent is increasedby about 2-fold to about 50-fold, by about 2-fold to about 25-fold, byabout 2-fold to about 20-fold, by about 2-fold to about 15-fold, byabout 2-fold to about 10-fold, by about 2-fold to about 5-fold, by about5-fold to about 25-fold, by about 5-fold to about 20-fold, by about5-fold to about 15-fold, by about 5-fold to about 10-fold, by about10-fold to about 25-fold, or by about 1-fold to about 20-fold per day ascompared to the release rate of the antimicrobial agent from anintravaginal ring comprising the antimicrobial agent without dapivirine.

In one embodiment, the cumulative release rate of the antimicrobialagent from an intravaginal ring of the invention comprising dapivirineand the antimicrobial agent is increased at least about 2-fold after 30days, after 60 days, or after 90 days as compared to the cumulativerelease rate of the antimicrobial agent from an intravaginal ringcomprising the antimicrobial agent without dapivirine after 30 days,after 60 days, or after 90 days. In one embodiment, the cumulativerelease rate of the antimicrobial agent from an intravaginal ring of theinvention comprising dapivirine and the antimicrobial agent is increasedat least about 10-fold after 30 days, after 60 days, or after 90 days ascompared to the cumulative release rate of the antimicrobial agent froman intravaginal ring comprising the antimicrobial agent withoutdapivirine after 30 days, after 60 days, or after 90 days. In anotherembodiment, the cumulative release rate of the antimicrobial agent froman intravaginal ring of the invention comprising dapivirine and theantimicrobial agent is increased at least about 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 35, 40, 45 or 50-fold after 30 days, after 60 days, or after 90days as compared to the cumulative release rate of the antimicrobialagent from an intravaginal ring comprising the antimicrobial agentwithout dapivirine after 30 days, after 60 days, or after 90 days. Inone embodiment, the cumulative release rate of the antimicrobial agentfrom an intravaginal ring of the invention comprising dapivirine and theantimicrobial agent is increased by about 2-fold to about 50-fold, byabout 2-fold to about 25-fold, by about 2-fold to about 20-fold, byabout 2-fold to about 15-fold, by about 2-fold to about 10-fold, byabout 2-fold to about 5-fold, by about 5-fold to about 25-fold, by about5-fold to about 20-fold, by about 5-fold to about 15-fold, by about5-fold to about 10-fold, by about 10-fold to about 25-fold, or by about1-fold to about 20-fold after 30 days, after 60 days, or after 90 daysas compared to the cumulative release rate of the antimicrobial agentfrom an intravaginal ring comprising the antimicrobial agent withoutdapivirine after 30 days, after 60 days, or after 90 days.

In one embodiment, the release rate of the dapivirine from anintravaginal ring comprising dapivirine and the antimicrobial compoundis not increased as compared to the release rate of dapivirine from anintravaginal ring comprising only dapivirine. In another embodiment, therelease rate of the dapivirine from an intravaginal ring comprisingdapivirine and the antimicrobial compound is also increased as comparedto the release rate of dapivirine from an intravaginal ring comprisingonly dapivirine.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

IV. Contraceptives

The instant invention is based on the surprising discovery thatintravaginal rings comprising dapivirine and a contraceptive result in asynergetic composition with enhanced release rates of either thecontraceptive or dapivirine, as compared to release rates of thecompounds, alone.

As used herein, the term “contraceptive” refers to an active agent thatprevents conception or pregnancy. Contraceptives are well-known in theart and include, but are not limited to, steroid hormones and include,for example, an estrogen, a progestin, a progesterone, a testosterone,derivatives thereof, or combinations thereof. Examples of contraceptivesinclude 17a-ethinyl-levonorgestrel-17b-hydroxy-estra-4,9,11-trien-3-one,estradiol, etonogestrel, levonorgestrel, medroxyprogesterone acetate,nestorone, norethindrone, and progesterone. In one embodiment of theinvention, the contraceptive is levonorgestrel. In one embodiment of theinvention, the contraceptive is estradiol. In one embodiment of theinvention, the contraceptive is etonorgestrel. In one embodiment of theinvention, the contraceptive is nestorone.

As used herein, an “estrogen” refers to any of various natural orsynthetic compounds that stimulate the development of female secondarysex characteristics and promote the growth and maintenance of the femalereproductive system, or any other compound that mimics the physiologicaleffect of natural estrogens. Estrogens suitable for use with the presentinvention also include compounds that can be converted to activeestrogenic compounds. For example, in some embodiments, a conjugatedestrogen can be administered from an intravaginal device of the presentinvention. As used herein, the term “conjugated” refers to the sulfateester, glucuronide ester, or mixed sulfate-glucuronide esters, of anestrogen. Estrogens suitable for use with the present invention alsoinclude pharmaceutically suitable salt forms of estrogens. In someembodiments, the salt can be a sodium, potassium, or2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) salt. An estrogensuitable for use with the present invention can be useful as acontraceptive agent.

Estrogens suitable for use in the present invention include, but are notlimited to, natural and synthetic compounds having estrogenic activity,such as, for example, estradiol (17β-estradiol), 17α-estradiol, estriol,estrone, and their esters, such as the acetate, sulfate, valerate orbenzoate esters of these compounds, including, for example, estradiol17β-cypionate, estradiol 17-propionate, estradiol 3-benzoate, andpiperazine estrone sulfate; ethinyl estradiol; conjugated estrogens(natural and synthetic); agonistic anti-estrogens; and selectiveestrogen receptor modulators.

Prodrugs of suitable estrogens can also be used in the device of thepresent invention. As used herein, a “prodrug” denotes a derivative of aknown direct acting drug, which derivative has enhanced deliverycharacteristics and therapeutic value as compared to the drug and istransformed into the active drug by an enzymatic or chemical process.Examples of estrogen prodrugs include, but are not limited to, estradiolacetate (which is converted in vivo to 17β-estradiol) and mestranol(which is converted in vivo to ethinyl estradiol). In some embodiments,the estrogen is estradiol, estriol, mestranol, ethinyl estradiol,diethylstilbestrol, or combinations thereof.

As used herein, a “progestin” refers to a progestogen, a progestationalsubstance, or any pharmaceutically acceptable substance in the steroidart that generally possesses progestational activity including syntheticsteroids that have progestational activity. Progestins suitable for usewith the present invention can be of natural or synthetic origin.Progestins generally possess a cyclo-pentanophertanthrene nucleus.

Progestins suitable for use in the present invention include, but arenot limited to, natural and synthetic compounds having progestationalactivity, such as, for example, progesterone, medroxyprogesterone,medroxyprogesterone acetate, chlormadinone acetate, norethindrone,cyproterone acetate, norethindrone acetate, desogestrel, levonorgestrel,drospirenone, trimegestone, norgestrel, norgestimate, norelgestromin,etonogestrel, dienogest, gestodene, megestrol, and other natural and/orsynthetic gestagens. In some embodiments, the progestin is progesterone,etonogestrel, levonorgestrel, gestodene, norethisterone, drospirenone,or combinations thereof. In one embodiment, the progestin islevonorgestrel. In another embodiment, the progestin is nesterone.

Prodrugs of suitable progestins can also be used in the intravaginaldevice of the present invention. Ethynodiol diacetate, which isconverted in vivo to norethindrone, is an example of a progestin prodrugthat can be used in the present invention. Additional examples ofprogestin prodrugs include, but are not limited to, norgestimate (whichis converted in vivo to 17-deacetyl norgestimate, also known asnorelgestromin), desogestrel (which is converted in vivo to 3-ketodesogestrel, also known as etonogestrel), and norethindrone acetate(which is converted in vivo to norethindrone).

In some embodiments, the progestin is desogestrel, etonogestrel,norgestimate, or combinations thereof.

In some embodiments, the active agent is a modified testosterone, e.g.,a derivative of the synthetic steroid ethisterone. In some embodiments,the modified testosterone is danazol.

In some embodiments, the intravaginal ring of the present invention maycomprise dapivirine and two contraceptive agents, such as a progestinand an estrogen. In some embodiments, the intravaginal ring comprisesdapivirine and a combination of a progestin and an estrogen.

In one embodiment, about 10 to about 30 mg of contraceptive is presentin the ring. In another embodiment, about 20 mg to about 30 mg ofcontraceptive is present in the ring. In yet another embodiment, about10 to about 800 mg, about 50 mg to about 750 mg, about 100 mg to about700 mg, or about 200 mg to about 600 mg, about 300 mg to about 400 mg ofcontraceptive is present in the ring.

In another embodiment, about 15 mg of contraceptive is present in thering. In another embodiment, about 16 mg of contraceptive is present inthe ring. In another embodiment, about 25 mg of contraceptive is presentin the ring. In another embodiment, about 32 mg of contraceptive ispresent in the ring. In another embodiment, about 100 mg ofcontraceptive is present in the ring. In another embodiment, about 150mg of contraceptive is present in the ring. In another embodiment, about250 mg of contraceptive is present in the ring. In another embodiment,about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg,about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, about16 mg, about 17 mg, about 18 mg, about 19 mg, about 20 mg, about 21 mg,about 22 mg, about 23 mg, about 24 mg, about 25 mg, about 26 mg, about27 mg, about 28 mg, about 29 mg, about 30 mg, about 31 mg, about 32 mg,about 33 mg, about 34 mg, about 35 mg, about 36 mg, about 37 mg, about38 mg, about 39 mg, about 40 mg, about 41 mg, about 42 mg, about 43 mg,about 44 mg, about 45 mg, about 46 mg, about 47 mg, about 48 mg, about49 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg,about 75 mg, about 80 mg, about 90 mg, about 100 mg, about 125 mg, about150 mg, about 175 mg, about 200 mg, about 225 mg, about 250 mg, about275 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about750 mg, or about 800 mg of contraceptive is present in the ring.

Surprisingly, the combination of dapivirine and a contraceptive, e.g.,levonorgestrel, in the intravaginal rings of the invention results inthe formation of a eutectic composition and provides an increasedrelease rate of the contraceptive, e.g., levonorgestrel, as compared tothe release rate of the contraceptive, e.g., levonorgestrel, inintravaginal rings comprising the contraceptive, e.g., levonorgestrel,without dapivirine. For example, in one embodiment, the release rate ofthe contraceptive from an intravaginal ring of the invention comprisingdapivirine and the contraceptive is increased at least about 2-fold perday as compared to the release rate of the contraceptive from anintravaginal ring comprising the contraceptive without dapivirine. Inone embodiment, the release rate of the contraceptive from anintravaginal ring of the invention comprising dapivirine and thecontraceptive is increased at least about 10-fold per day as compared tothe release rate of the contraceptive from an intravaginal ringcomprising the contraceptive without dapivirine. In another embodiment,the release rate of the contraceptive from an intravaginal ring of theinvention comprising dapivirine and the contraceptive is increased atleast about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50-fold perday as compared to the release rate of the contraceptive from anintravaginal ring comprising the contraceptive without dapivirine. Inone embodiment, the release rate of the contraceptive from anintravaginal ring of the invention comprising dapivirine and thecontraceptive is increased by about 2-fold to about 50-fold, by about2-fold to about 25-fold, by about 2-fold to about 20-fold, by about2-fold to about 15-fold, by about 2-fold to about 10-fold, by about2-fold to about 5-fold, by about 5-fold to about 25-fold, by about5-fold to about 20-fold, by about 5-fold to about 15-fold, by about5-fold to about 10-fold, by about 10-fold to about 25-fold, or by about1-fold to about 20-fold per day as compared to the release rate of thecontraceptive from an intravaginal ring comprising the contraceptivewithout dapivirine.

In one embodiment, the cumulative release rate of the contraceptive froman intravaginal ring of the invention comprising dapivirine and thecontraceptive is increased at least about 2-fold after 30 days, after 60days, or after 90 days as compared to the cumulative release rate of thecontraceptive from an intravaginal ring comprising the contraceptivewithout dapivirine after 30 days, after 60 days, or after 90 days. Inone embodiment, the cumulative release rate of the contraceptive from anintravaginal ring of the invention comprising dapivirine and thecontraceptive is increased at least about 10-fold after 30 days, after60 days, or after 90 days as compared to the cumulative release rate ofthe contraceptive from an intravaginal ring comprising the contraceptivewithout dapivirine after 30 days, after 60 days, or after 90 days. Inanother embodiment, the cumulative release rate of the contraceptivefrom an intravaginal ring of the invention comprising dapivirine and thecontraceptive is increased at least about 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 35, 40, 45 or 50-fold after 30 days, after 60 days, or after 90 daysas compared to the cumulative release rate of the contraceptive from anintravaginal ring comprising the contraceptive without dapivirine after30 days, after 60 days, or after 90 days. In one embodiment, thecumulative release rate of the contraceptive from an intravaginal ringof the invention comprising dapivirine and the contraceptive isincreased by about 2-fold to about 50-fold, by about 2-fold to about25-fold, by about 2-fold to about 20-fold, by about 2-fold to about15-fold, by about 2-fold to about 10-fold, by about 2-fold to about5-fold, by about 5-fold to about 25-fold, by about 5-fold to about20-fold, by about 5-fold to about 15-fold, by about 5-fold to about10-fold, by about 10-fold to about 25-fold, or by about 1-fold to about20-fold after 30 days, after 60 days, or after 90 days as compared tothe cumulative release rate of the contraceptive from an intravaginalring comprising the contraceptive without dapivirine after 30 days,after 60 days, or after 90 days.

In one embodiment, the release rate of the dapivirine from anintravaginal ring of the invention comprising dapivirine and acontraceptive is not increased as compared to the release rate ofdapivirine from an intravaginal ring comprising only dapivirine. Inanother embodiment, the release rate of the dapivirine from anintravaginal ring of the invention comprising dapivirine and acontraceptive is also increased as compared to the release rate ofdapivirine from an intravaginal ring comprising only dapivirine.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

V. Methods for Preventing/Treating HIV and/or Pregnancy

The present invention provides methods of preventing and/or treating HIVand preventing pregnancy using the intravaginal rings of the inventioncomprising dapivirine and a contraceptive. The present invention alsoprovides methods of preventing and/or treating HIV using theintravaginal rings of the invention comprising dapivirine and anantimicrobial compound. In one aspect, the present invention providesmethods of blocking DNA polymerization by an HIV reverse transcriptaseenzyme in a female human, comprising the step of inserting anintravaginal ring of the invention into the vagina of the female human.In another aspect, the present invention provides methods of preventingHIV infection in a female human, comprising the step of inserting anintravaginal ring of the invention into the vagina of the female human.In yet another aspect, the invention provides methods of treating HIVinfection in a female human, comprising the step of inserting anintravaginal ring of the invention into the vagina of the female human.In another aspect, the invention provides methods of preventingpregnancy in a female human, comprising the step of inserting anintravaginal ring of the invention into the vagina of the female human.

The ring that is inserted into a human may contain a prophylacticallyeffective amount or a therapeutically effective amount of dapivirine andan antimicrobial compound, e.g., maraviroc. The ring that is insertedinto a human may contain a prophylactically effective amount or atherapeutically effective amount of dapivirine and a contraceptive,e.g., levonorgestrel.

As used herein, the term “prophylactically effective amount” refers tothe amount of dapivirine or the amount of antimicrobial compoundeffective to prevent development of disease in the subject. In oneembodiment of the invention, the disease is HIV. In a preferredembodiment of the invention, a prophylactically effective amount isachieved when less than about 7 mg, or when between about 1 mg and about3 mg of dapivirine and/or antimicrobial compound is released in vitroduring an initial 24 hour period of release. In one embodiment of theinvention, a prophylactically effective amount is achieved when betweenabout 100 μg to about 700 μg, or when between about 100 mg to about 700mg of dapivirine and/or antimicrobial compound is released in vitro perday after the initial 7 day period of release. In one embodiment of theinvention, a prophylactically effective amount is achieved when betweenabout 200 μg to about 400 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In another embodiment of the invention, a prophylactically effectiveamount is achieved when between about 250 μg to about 350 μg ofdapivirine and/or antimicrobial compound is released in vitro per dayafter the initial 7 day period of release. In another embodiment of theinvention, a prophylactically effective amount is achieved when betweenabout 300 μg to about 400 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In one embodiment, a prophylactically effective amount is achieved whenabout 50 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg,about 200 μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg,about 325 μg, about 350 μg, about 375 μg, about 400 μg, about 450 μg,about 475 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg,about 700 μg, about 750 μg, about 800 μg, about 850 μg, about 900 μgabout 950 μg, about 1 mg, less than 1 mg or more than 1 mg of dapivirineand/or antimicrobial compound is released in vitro per day after theinitial 7 day period of release.

In another embodiment, a prophylactically effective amount is achievedwhen between about 5 μg and about 300 μg of dapivirine and/orantimicrobial compound is released in vivo per gram of vaginal fluideach day for 24 days after an initial 3 day period of use. In anotherembodiment, a prophylactically effective amount is achieved when betweenabout 10 μg and about 100 μg of dapivirine and/or antimicrobial compoundis released in vivo per gram of vaginal fluid each day for 24 days afteran initial 3 day period of use. In another embodiment, aprophylactically effective amount is achieved when between about 20 μgand about 80 μg of dapivirine and/or antimicrobial compound is releasedin vivo per gram of vaginal fluid each day for 24 days after an initial3 day period of use. In yet another embodiment, a prophylacticallyeffective amount is achieved when about 5 μg, about 10 μg, about 25 μg,about 50 μg, about 75 μg, about 100 μg, about 125 μg, about 150 μg,about 175 μg, about 200 μg, about 225 μg, about 250 μg, about 275 μg,about 300 μg, about 350 μg, about 400 μg, about 450 μg, about 500 μg, orabout 600 μg of dapivirine and/or antimicrobial compound is released invivo per gram of vaginal fluid each day for 24 days after an initial 3day period of use.

As used herein, the term “prophylactically effective amount” refers tothe amount of contraceptive effective to prevent contraception orpregnancy in the subject. In one embodiment of the invention, aprophylactically effective amount is achieved when less than about 2 gof the contraceptive is released in vitro during an initial 24 hourperiod of release. In another embodiment, a prophylactically effectiveamount is achieved when less than about 1 g of the contraceptive isreleased in vitro during an initial 24 hour period of release. In oneembodiment of the invention, a prophylactically effective amount isachieved when about 20 μg per day to about 290 μg per day of thecontraceptive is released in vitro for about 23 days after the initial 7day period of release. In one embodiment of the invention, aprophylactically effective amount is achieved when about 20 μg per dayto about 290 μg per day of the contraceptive is released in vitro forabout 53 days after the initial 7 day period of release. In oneembodiment of the invention, a prophylactically effective amount isachieved when about 35 μg per day to about 70 μg per day of thecontraceptive is released in vitro for about 23 days after the initial 7day period of release. In one embodiment of the invention, aprophylactically effective amount is achieved when about 35 μg per dayto about 70 μg per day of the contraceptive is released in vitro forabout 53 days after the initial 7 day period of release.

In one embodiment of the invention, a prophylactically effective amountis achieved when less than about 100 μg per day of the contraceptive isreleased in vitro after the initial 7 day period of release for about 23days. In one embodiment of the invention, a prophylactically effectiveamount is achieved when less than about 100 μg per day of thecontraceptive is released in vitro after the initial 7 day period ofrelease for about 53 days. In one embodiment of the invention, aprophylactically effective amount is achieved when less than about 70 μgper day of the contraceptive is released in vitro after the initial 7day period of release for about 23 days. In one embodiment of theinvention, a prophylactically effective amount is achieved when lessthan about 70 μg per day of the contraceptive is released in vitro afterthe initial 7 day period of release for about 53 days. In one embodimentof the invention, a prophylactically effective amount is achieved whenless than about 15 μg per day of the contraceptive is released in vitroafter the initial 7 day period of release for about 23 days. In oneembodiment of the invention, a prophylactically effective amount isachieved when less than about 15 μg per day of the contraceptive isreleased in vitro after the initial 7 day period of release for about 53days. In one embodiment of the invention, a prophylactically effectiveamount is achieved when about 35 μg per day of the contraceptive isreleased in vitro after the initial 7 day period of release for about 23days or for about 53 days.

In one embodiment of the invention, a prophylactically effective amountis achieved when about 20 μg per day to about 290 μg per day of thecontraceptive is released in vitro for about 30 days. In one embodimentof the invention, a prophylactically effective amount is achieved whenabout 20 μg per day to about 290 μg per day of the contraceptive isreleased in vitro for about 60 days. In one embodiment of the invention,a prophylactically effective amount is achieved when about 35 μg per dayto about 70 μg per day of the contraceptive is released in vitro forabout 30 days. In one embodiment of the invention, a prophylacticallyeffective amount is achieved when about 35 μg per day to about 70 μg perday of the contraceptive is released in vitro for about 60 days.

In one embodiment of the invention, a prophylactically effective amountis achieved when less than about 100 μg per day of the contraceptive isreleased in vitro for about 30 days. In one embodiment of the invention,a prophylactically effective amount is achieved when less than about 100μg per day of the contraceptive is released in vitro for about 60 days.In one embodiment of the invention, a prophylactically effective amountis achieved when less than about 70 μg per day of the contraceptive isreleased in vitro for about 30 days. In one embodiment of the invention,a prophylactically effective amount is achieved when less than about 70μg per day of the contraceptive is released in vitro for about 60 days.In one embodiment of the invention, a prophylactically effective amountis achieved when less than about 35 μg per day of the contraceptive isreleased in vitro for about 30 days. In one embodiment of the invention,a prophylactically effective amount is achieved when less than about 35μg per day of the contraceptive is released in vitro for about 60 days.In one embodiment of the invention, a prophylactically effective amountis achieved when less than about 15 μg per day of the contraceptive isreleased in vitro for about 30 days. In one embodiment of the invention,a prophylactically effective amount is achieved when less than about 15μg per day of the contraceptive is released in vitro for about 60 days.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

As used herein, the term “therapeutically effective amount” refers tothe amount of compound effective to treat disease in the subject. In oneembodiment of the invention, the disease is HIV. In a preferredembodiment of the invention, a therapeutically effective amount isachieved when less than about 7 mg, or between about 1 mg and about 3 mgof dapivirine and/or antimicrobial compound is released in vitro duringan initial 24 hour period of release. In one embodiment of theinvention, a therapeutically effective amount is achieved when betweenabout 100 μg to about 700 μg, or between about 100 mg and about 500 mgof dapivirine and/or antimicrobial compound is released in vitro per dayafter the initial 7 day period of release. In one embodiment of theinvention, a therapeutically effective amount is achieved when betweenabout 200 μg to about 400 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In another embodiment of the invention, a therapeutically effectiveamount is achieved when between about 250 μg to about 350 μg ofdapivirine and/or antimicrobial compound is released in vitro per dayafter the initial 7 day period of release. In another embodiment of theinvention, a therapeutically effective amount is achieved when betweenabout 100 μg to about 400 μg of dapivirine and/or antimicrobial compoundis released in vitro per day after the initial 7 day period of release.In one embodiment, a therapeutically effective amount is achieved whenabout 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200 μg,about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 325 μg,about 350 μg, about 375 μg, about 400 μg, about 450 μg, about 475 μg,about 500 μg, about 550 μg, about 600 μg, about 650 μg, or about 700 μgof dapivirine and/or antimicrobial compound is released in vitro per dayafter the initial 7 day period of release.

In another embodiment, a therapeutically effective amount is achievedwhen between about 5 μg and about 300 μg of dapivirine and/orantimicrobial compound is released in vivo per gram of vaginal fluideach day for 24 days after an initial 3 day period of use. In anotherembodiment, a therapeutically effective amount is achieved when betweenabout 10 μg and about 100 μg of dapivirine and/or antimicrobial compoundis released in vivo per gram of vaginal fluid each day for 24 days afteran initial 3 day period of use. In another embodiment, a therapeuticallyeffective amount is achieved when between about 20 μg and about 80 μg ofdapivirine and/or antimicrobial compound is released in vivo per gram ofvaginal fluid each day for 24 days after an initial 3 day period of use.In yet another embodiment, a therapeutically effective amount isachieved when about 5 μg, about 10 μg, about 25 μg, about 50 μg, about75 μg, about 100 μg, about 125 μg, about 150 μg, about 175 μg, about 200μg, about 225 μg, about 250 μg, about 275 μg, about 300 μg, about 350μg, about 400 μg, about 450 μg, about 500 μg, or about 600 μg ofdapivirine and/or antimicrobial compound is released in vivo per gram ofvaginal fluid each day for 24 days after an initial 3 day period of use.

Values and ranges included and/or intermediate within the ranges setforth herein are also intended to be within the scope of the presentdisclosure. Ranges having values recited herein as an upper or lowerlimit are also intended to be within the scope of the presentdisclosure.

Those of skill in the prevention and/or treatment of HIV and theprevention of pregnancy could determine the appropriate therapeuticallyeffective amount or prophylactically effective amount from the datapresented here in the Examples section. The exact dosage may depend onthe particular active agent used.

The term “subject” means female humans who use the rings. Administrationof the rings of the present invention to a subject can be carried outusing known procedures, at dosages and for periods of time effective totreat or prevent HIV or to prevent pregnancy.

As used herein, the term “vagina” or “vaginal” refers to the passageleading from the opening of the vulva to the cervix of the uterus infemale humans. As used herein, the term “intravaginal administering”refers to the administration of a ring of the invention to the vagina ofa female human.

The rings of the present invention may be administered into the vaginaof a subject prior to sexual intercourse, e.g., 1, 2, 3, 4, 5 or 6weeks, prior to sexual intercourse. In some embodiments, the rings ofthe invention may be administered into the vagina of a subject aftersexual intercourse, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14or 15 days after sexual intercourse.

The term sexual intercourse means vaginal sex.

The term “partners” as used herein defines two or more humans, who aresexually active with each other, i.e., who have sexual intercourse witheach other.

As used herein, the term “preventing HIV infection” or “preventing HIVtransmission” includes the application or administration of anintravaginal ring of the invention to a subject who is at risk ofdeveloping HIV, or who has been exposed to but not yet developed HIV, inorder to decrease the likelihood that the subject will develop HIV. Inone embodiment of the invention, the term “preventing HIV infection”includes the application or administration of an intravaginal ring ofthe invention to a subject who is at risk of developing HIV, or who hasbeen exposed to but not yet developed HIV, in order to decrease thelikelihood that the subject will develop HIV, as compared to a subjectwho has not been administered an intravaginal ring. In one embodiment ofthe invention, proper use of the intravaginal rings of the inventionleads to prevention of HIV infection in about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or about 100% of the subjects whoare at risk of developing HIV or who have been exposed to but not yetdeveloped HIV. Values and ranges included and/or intermediate within theranges set forth herein are also intended to be within the scope of thepresent disclosure. Ranges having values recited herein as an upper orlower limit are also intended to be within the scope of the presentdisclosure.

As used herein, the term “preventing pregnancy” includes the applicationor administration of an intravaginal ring of the invention to a subjectwho is at risk of becoming pregnant in order to decrease the likelihoodthat the subject will become pregnant. In one embodiment of theinvention, the term “preventing pregnancy” includes the application oradministration of an intravaginal ring of the invention to a subject whois at risk of becoming pregnant in order to decrease the likelihood thatthe subject will become pregnant, as compared to a subject who has notbeen administered an intravaginal ring. In one embodiment of theinvention, proper use of the intravaginal rings of the invention leadsto prevention of pregnancy infection in about 1%, about 5%, about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, about 50%, about 55%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99% or about 100% of the subjects whoare at risk of becoming pregnant. Values and ranges included and/orintermediate within the ranges set forth herein are also intended to bewithin the scope of the present disclosure. Ranges having values recitedherein as an upper or lower limit are also intended to be within thescope of the present disclosure.

The term “treating” includes the application or administration of anintravaginal ring of the invention to a subject, or application oradministration of an intravaginal ring of the invention to a subject whohas HIV, with the purpose of curing, healing, alleviating, relieving,altering, remedying, ameliorating, preventing, improving, or affectingHIV. The term “treating” refers to any indicia of success in thetreatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; diminishing of symptoms or making the injury, pathology orcondition more tolerable to the subject; slowing in the rate ofdegeneration or decline; making the final point of degeneration lessdebilitating; or improving a subject's physical or mental well-being.Treatment may be therapeutic or prophylactic. The treatment oramelioration of symptoms can be based on objective or subjectiveparameters; including the results of a physical examination.

VI. Methods for Preparing Rings of the Invention

Rings of the invention may be manufactured by any method known by thoseskilled-in-the-art, but preferably by injection molding or extrusion,and more preferably by reaction injection molding of silicone elastomersystems. The term ‘injection molding’ refers to manufacturing processesfor producing parts/devices from either thermoplastic or thermosettingmaterials using suitably designed injection molds. Examples ofthermoplastic materials include polyethylene and PEVA; examples ofthermosetting materials include silicone rubbers/elastomers. Withoutlimitation, matrix-type silicone elastomer rings containing dapivirinemay be prepared by (i) adding and mixing the dapivirine into one or morecomponents of the silicone system (e.g., base, crosslinking agent,catalyst, excipient, dispersant, etc) (ii) injecting the mix intosuitably designed injection molds, and (iii) optionally, applying heatto cause the silicone mix to cure/crosslink forming an elastomer.

The present invention further provides methods of preparing theintravaginal rings of the invention described above. These methodsgenerally comprise dispersing dapivirine and either an antimicrobialagent or a contraceptive, and an elastomer, e.g., polysiloxane, in anappropriate solvent or dispersing agent, e.g., silicone liquid, andcuring the rings with a platinum catalyst, e.g., a platinum-siloxanecomplex, thereby preparing a platinum-catalyzed ring. Any of thewell-known elastomers, e.g., polysiloxanes, described supra may be usedto prepare the platinum-catalyzed rings of the invention. In oneembodiment, an elastomer, e.g., polysiloxane, for use in the methods ofthe invention is a dimethylsiloxane, e.g., vinyl-terminatedpolydimethylsiloxane. In another embodiment, an elastomer, e.g.,polysiloxane, for use in the methods of the invention is adiorganopopolysiloxane, e.g., dimethylpolysiloxane. In anotherembodiment, the elastomer, e.g., polysiloxane, for use in the methods ofthe invention is MED-8470. In certain embodiments, the methods furthercomprise use of a cross-linker, e.g., hydride functionalpolydimethylsiloxane or dimethylmethylhydrogen polysiloxane cross-link.

In one embodiment, the method further comprises catalyzing the rings ina ring mould. The mould can then be opened, following which theintravaginal ring is removed and trimmed. Ring moulds, are preferablycoated with, for example, Teflon™ or an electrolytically appliedmetalised coating. Ring moulds may be constructed of hardened carbonsteel, stainless steel, aluminum, or any other material deemed to beappropriate. It will be appreciated that the mould dimensions and designimpart the physical shape of the intravaginal drug delivery device, forexample, a partial or complete ring, or any other desired shape.Preferably, the device has a partial or complete toroidal shape, morepreferably a partial or complete torus shape, or a substantiallycylindrical shape. By toroid is meant a ring-like body generated byrotating any closed loop (including an ellipse, a circle or anyirregular curve) about a fixed line external to that loop. The toroidshape may be a complete or partial toroid. By torus is meant a ring-likebody generated by rotating a circle about a fixed line external to thecircle. The torus shape may be a complete or partial ring-like shape.The geometric characteristics of the mould and intravaginal rings can bevaried as required by the use.

Alternatively, the intravaginal ring device, or components thereof, maybe prepared by extrusional processes, e.g., co-extrusion or blendextrusion, well known to those skilled in the art (see, e.g., U.S. Pat.No. 5,059,363, the entire contents of which are incorporated herein byreference).

The present invention is further illustrated by the following examples,which should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application, as well as the Figures, are expresslyincorporated herein by reference in their entirety.

EXAMPLES Example 1: Eleven Prototype Rings in MED4870 ComprisingDapivirine and a Contraceptive

The purpose of this work was to establish whether a matrix-type ringcontaining both dapivirine (DAP) and a contraceptive, such aslevonorgestrel (LNG), might be a suitable formulation for a product,i.e., a ring that prevents HIV transmission and also provides effectivecontraceptive cover over a period of 30 days or 60 days.

Three loadings of DAP were chosen for further investigation in thisstudy: 100 mg, 150 mg and 200 mg. LNG loadings of 16 mg and 32 mg wereselected as being likely to provide clinically meaningful in vitrorelease rates over 60 days. Various combinations of the two APIs atthese loadings were also included in the study, with eleven prototypeformulations manufactured in total (see Table 1). Testing offormulations that continued to provide release of DAP and LNG at levelshigher than the minimum acceptable level at day 60 was continued out toa total of 92 days.

A Babyplast injection molder and a mold set of cross-sectional diameter7.82 mm, outer diameter 56.67 mm were used to manufacture the rings. Thesilicone elastomer used was MED-4870.

Dapivirine (micronised) was supplied by IPM. Levonorgestrel(non-micronised) was supplied by Haorui Pharma-Chem Inc. (Irvine,Calif.). MED-4870 silicone elastomer was obtained from NuSil. HPLC-gradeacetonitrile, HPLC-grade isopropanol and potassium dihydrogenorthophosphate (AnalR analytical reagent) were purchased from VWRInternational Ltd. (Dublin, Ireland). Phosphoric acid (85% w/w in water)was purchased from Sigma-Aldrich (Gillingham, UK). A Millipore Direct-Q3 UV Ultrapure Water System (Watford, UK) was used to obtain HPLC-gradewater.

TABLE 1 Eleven matrix-type ring prototype formulations DAP DAP LNG LNGFormulation Loading Loading Loading Loading Code (mg/ring) % w/w mg/ring% w/w A 100 1.25 — — B 150 1.875 — — C 200 2.5 — — D — — 16 0.2 E — — 320.4 F 100 1.25 16 0.2 G 100 1.25 32 0.4 H 150 1.875 16 0.2 I 150 1.87532 0.4 J 200 2.5 16 0.2 K 200 2.5 32 0.4

Premixes were first prepared by mixing appropriate quantities of eachAPI with Part A or Part B of the silicone elastomer (Table 2). For eachformulation, Premix A and Premix B were prepared in a 100 g batch size.The steps for preparation of 100 g of each premix were as follows: 1)Quantity of API (DAP and/or LNG) weighed into SpeedMixer tub accordingto Table 2. 2) Quantity of MED-4870 (Part A or Part B) added toSpeedMixer tub according to Table 2. 3) SpeedMixer tub sealed andcontents mixed using SpeedMixer (3 min at 3000 rpm). 4) Premixes storedin fridge until required for injection moulding. 5) On date ofmanufacture, premixes were removed from fridge, hand mixed for 30 s andthen SpeedMixed for 120 s @ 3000 rpm.

TABLE 2 Quantities of each component in 100 g premix Mass of Mass ofMass of MED-4870 Part A Formulation DAP (g) LNG (g) or Part B (g) A 1.25— 98.75 B 1.8758 — 98.125 C 2.5 — 97.5 D — 0.2 99.8 E — 0.4 99.6 F 1.250.2 99.55 G 1.25 0.4 98.35 H 1.875 0.2 97.925 I 1.875 0.4 97.725 J 2.50.2 97.3 K 2.5 0.4 97.1

Prior to injection moulding of rings, premixes of A and B were combinedin a 1:1 ratio, according to the following procedure:

1. Premix A and 50 g of Premix B were added to the SpeedMixer tub inlayers (25 g Premix A, then 25 g Premix B, then 25 g Premix A, then 25 gPremix B) and hand mixed for 30 s.

2. Tub placed into SpeedMixer (30 s at 3000 rpm).

3. Process repeated for each formulation until 4×100 g total A/B mixtureproduced.

4. Contents of each tub were transferred to a 500 g cartridge thatoperated with the dosing system on the Babyplast injection molder.

Heating of the ring mold assembly on the Babyplast machine was performedvia 2×200 W heater cartridges on both the fixed and mobile plates.Injection parameters were as follows: Clamping pressure: 100 bar,Injection pressure: 50 bar, Mould temperature: 160° C., Cure time: 60 s.The measured dimensions of the ring mold were as follows: outerdiameter: 56.67 mm, cross-sectional diameter: 7.82 mm.

In vitro release testing was performed daily (excluding weekends) forall rings over 30 days, followed by twice weekly sampling through day60. Selected formulations (C and K) were tested twice weekly up to day92. On day 0, each ring was placed into a 250 mL glass bottle containing200 mL dissolution medium (1:1 mixture of isopropanol and water) andstored in an orbital shaking incubator (37° C., 60 rpm, 25 mm orbitalthrow). The dissolution medium was sampled and replaced (100 mL) dailywith the exception of weekends (200 mL volume over weekend) until day30. On day 30, 200 mL release medium was added to each bottle. The nextsample time-point was day 38, and the release medium was sampled andcompletely replaced (100 mL). Samples were taken again on day 39 and therelease medium was replaced with 200 mL. The same protocol was followedon days 45 and 46, days 52 and 53, and days 59 and 60. On the basis ofobserved release at day 60, release testing was extended to day 92 forformulations C and K; sampling was conducted on days 66 and 67, 73 and74, 80 and 81, 87 and 88, 91 and 92 according to the same protocol. Drugrelease was quantified by reverse-phase HPLC analysis of all sampleswith UV detection.

The ‘multi-API HPLC method’ was used for analysis of all samplesobtained during the dissolution study. Briefly, 25 μL of each sample wasinjected onto a Thermo Scientific BDS Hypersil C18 column (150 mm×4.6mm, 3 μm particle size) fitted with a guard column. The column was heldat 25° C. and isocratic elution was performed using a mobile phase of55% 7.7 mM phosphate buffer (pH 3.0) and 45% HPLC-grade acetonitrile(1.2 mL/min) with a run time of 9 min. DAP was detected using awavelength of 210 nm after 6.2 min, while LNG was detected after 7.7 minusing a wavelength of 240 nm.

DAP and LNG release in vitro were compared for each ring set using aone-way ANOVA, followed by post-hoc analysis using the Tukey-Kramermultiple comparisons test. The following results were compared for bothdrugs: Day 1 release, Day 30 release, Day 60 release and Total releaseover 60 days. Analysis was conducted using GraphPad Prism software andsignificance was noted for a P value of less than 0.05: *=significant(0.01<P<0.05), **=very significant (0.001<P<0.01), ***=extremelysignificant (P<0.001), and ns=not significant (P >0.05).

Details of manufactured rings included in the in vitro release study arepresented in Table 3.

TABLE 3 Mean mass of rings used in the in vitro release study (n = 6 performulation) DAP loading LNG loading Target Mean Target Mean Mean ringDAP theoretical LNG theoretical Formulation mass (g) loading DAP loadingloading LNG loading Code (±sd) (mg) (mg) (±sd) (mg) (mg) (±sd) A 7.99(±0.01) 100  99.91 (±0.14) — — B 7.98 (±0.01) 150 149.66 (±0.22) — — C8.01 (±0.01) 200 200.20 (±0.25) — — D 7.98 (±0.01) — — 16 15.97 (±0.03)E 7.95 (±0.01) — — 32 31.82 (±0.02) F 7.97 (±0.01) 100  99.62 (±0.02) 1615.94 (±0.00) G 8.00 (±0.01) 100 100.02 (±0.07) 32 32.01 (±0.02) H 8.00(±0.01) 150 150.04 (±0.05) 16 16.00 (±0.00) I 8.00 (±0.01) 150 149.96(±0.08) 32 31.99 (±0.2)  J 7.97 (±0.01) 200 119.19 (±0.17) 16 15.94(±0.01) K 8.06 (±0.01) 200 201.53 (±0.17) 32 32.24 (±0.03)

Daily and cumulative release charts for DAP and LNG from each ring setare presented in FIGS. 1 and 2 respectively, up to and including day 60.A summary of all in vitro release data is presented in Tables 4 and 5.

TABLE 4 Summary of DAP release date for each formulation (n = 6). Day 30Day 60 Cumulative Daily Day 1 release release release after % Releaserelease R² (linear release (μg) (μg) (μg) 60 days after 60 rateregression Code (±sd) (±sd) (±sd) (mg) (±sd) days (±sd) (μg/day)analysis) A 4132 (±65)  414 (±2) 284 (±2) 36.7 (±0.2) 37 (±0.3) 47810.9993 B 5149 (±126) 514 (±6) 363 (±5)   45 (±0.2) 30 (±0.1) 5878 0.9997C 5951 (±90)  618 (±6) 437 (±1) 53.3 (±0.3) 27 (±0.2) 6975 0.9998 F 4149(±38)  409 (±4) 284 (±2) 36.1 (±0.1) 36 (±0.1) 4710 0.9994 G 4333 (±210)407 (±3) 285 (±2) 36.5 (±0.3) 37 (±0.3) 4741 0.9993 H 5360 (±280) 525(±4) 373 (±4) 46.0 (±0.1) 31 (±0.1) 5992 0.9997 I 5320 (±175) 531 (±5)376 (±2) 46.4 (±0.3) 31 (±0.2) 6056 0.9997 J 6113 (±60)  629 (±4) 449(±3) 54.2 (±0.1) 27 (±0.1) 7098 0.9999 K 6038 (±103) 634 (±5) 454 (±3)54.8 (±0.3) 27 (±0.1) 7173 0.9997

TABLE 5 Summary of LNG release date for each formulation (n = 6). Day 30Day 60 Cumulative Daily Day 1 release release release after % Releaserelease R² (linear release (μg) (μg) (μg) 60 days after 60 rateregression Code (±sd) (±sd) (±sd) (mg) (±sd) days (±sd) (μg/day)analysis) D 129 (±14)  6.5 (±3) 2.0 (±1) 0.89 (±0.03)  6 (±0.2) 1090.9503 E 303 (±35)  20 (±2) 5.2 (±1)  2.5 (±0.06)  8 (±0.2) 317 0.9347 F245 (±18)  64 (±2)  23 (±2)  5.0 (±0.09) 31 (±0.5) 757 0.9909 G 612(±61) 147 (±5)  84 (±4) 10.9 (±0.2)  34 (±0.6) 1608 0.9984 H 261 (±21) 69 (±2)  25 (±2)  5.1 (±0.07) 32 (±0.4) 781 0.9937 I 621 (±19) 158 (±2) 91 (±3) 11.6 (±0.1)  36 (±0.4) 1701 0.9984 J 286 (±13)  72 (±2)  29(±2)  5.4 (±0.09) 34 (±0.5) 819 0.9945 K 684 (±17) 155 (±4)  91 (±2)11.6 (±0.1)  36 (±0.4) 1696 0.9986

Formulations C and K were selected for extended in vitro releasetesting, up to day 92. Daily and cumulative release charts for DAP(formulations C and K) and LNG (formulation K only) are presented inFIGS. 3 and 4 respectively, up to and including day 92. A summary of theDAP and LNG release data over 92 days for formulations C and K isprovided in Table 6.

TABLE 6 DAP and LNG release data over 92 days for C and K (n = 6)Cumulative % release Release Day 92 after 92 after Daily R² (linearRelease days (mg) 92 days release regression Code API (μg)(±sd) (±sd)(μg/day) rate analysis) C DAP 301 (±23) 64.8 (±0.25) 32 (±0.1) 68520.9996 K DAP 299 (±5)  66.6 (±0.33) 33 (±0.2) 7032 0.9995 LNG 46 (±2)13.7 (±0.19) 43 (±0.6) 1595 0.9955

Release of DAP for each set of the DAP-loaded formulations followed thetypical profile expected for matrix rings, i.e., a high mass wasreleased on day 1 followed by steadily declining daily release over thecourse of the study (FIG. 1A). There is a linear relationship betweencumulative release and root time for all formulations (Table 4),indicating that DAP was released via a diffusion-controlled mechanism.

Increasing the DAP loading produced a significant increase in the DAPrelease rate (P<0.001 for all relevant comparisons). However, theadditional presence of LNG in the rings did not have a significantinfluence on DAP release. For rings containing 100 mg DAP and 0, 16 or32 mg LNG (i.e., formulations A, F and G), there was no significantdifference in DAP release for any of the comparisons made (P>0.05), withthe exception of total release from formulations A and F (P<0.01). Thetotal release of DAP from formulations A and F was 36.7 and 36.1 mgrespectively, so the difference may not be evident in vivo and maysimply be due to some minor inter-batch variation in the rings. The sameis true for rings containing 150 mg DAP (B, G and H) and 200 mg DAP (C,J and K); while some of the release data appears to be significantlydifferent, the actual values for release are very similar and may not beclinically significant. Alternatively, the presence of LNG could becausing a small increase in DAP release that would only become moreapparent if much higher loadings of LNG were present in the rings. Inall cases, low values of percentage RSD for the daily release data wereobserved, indicating that release is highly reproducible.

Based on in vitro release data generated, each formulation tested haspotential as a 60-day product. Day 60 DAP release was 284-285 μg for the100 mg DAP rings, 363-376 μg for the 150 mg DAP rings, and 437-454 μgfor the 200 mg DAP rings, all exceeding the Day 60 release target ofgreater than 200 μg.

LNG highest release occurred on day 1, followed by declining dailyrelease over time (FIG. 3A). LNG release increased with increasedinitial LNG loading (P<0.001 in all cases when equivalent formulationswith different LNG loadings are compared). Release wasdiffusion-controlled for at least part of the release period. A linearrelationship between cumulative release and root time was observed forformulations F-K over the full 60 days (i.e., DAP/LNG combinationrings), but only up to day 17 for formulations D and E (i.e., LNG-onlyrings) (Table 6). This suggests that the presence of DAP in the rings isinfluencing LNG release. LNG release is very significantly increased bythe presence of DAP when LNG-only rings are compared to DAP/LNG ringswith equivalent LNG loading (FIGS. 3A and 3B) (P<0.001 in all cases).For the LNG-only rings, release was almost unquantifiable by day 60,despite only a small fraction of the initial drug loading having beenreleased (6 and 8% for D and E, respectively). Each of the combinationrings still released significant quantities of LNG on day 60, with31-36% of the initial loading released over the course of the study(Table 6).

The presence of DAP in the ring may be enhancing the solubility of LNGin the silicone elastomer, resulting in a corresponding increase inrelease. Another possibility is that DAP and LNG are forming a eutecticmixture, which in turn is causing an increase in LNG release through areduction in the melting point of each drug. The data suggests that itis the co-presence of DAP in the ring, rather than the magnitude of theinitial loading, that causes a significant increase in LNG release. Inmost cases, a comparison of LNG release from rings containing the sameloading of LNG but varying loadings of DAP showed no significantdifference in release.

The LNG loadings for rings tested in this study were selected based ontarget day 60 release values for LNG of 35 μg and 70 μg. Formulations F,H and J all contained 16 mg LNG; release of LNG on day 60 was in therange 23-29 μg for each of these rings. This is slightly below the lowerLNG target of 35 μg/day on day 60, indicating that the loading may needto be increased to ensure final daily release is above this target. Ahigher LNG loading of 32 mg was studied in formulations G, I and K; day60 release from these rings ranged from 84 to 91 μg, i.e., above thehigher target of 70 μg.

Two ring formulations, C and K, were selected for extended in vitrorelease testing (out to day 92). Formulation C contained 200 mg DAP;formulation K contained 200 mg DAP and 32 mg LNG. Release of both DAPand LNG continued according to the same trends observed up to day 60,i.e., release of both APIs steadily decreased over the study (FIG. 3 )and there was a linear relationship between cumulative release and roottime over the entire 92 days (Table 6). The purpose of the extendedstudy was to determine whether a matrix ring might be suitable for useout to 3 months, as this would bring added benefits in terms of cost anduser-adherence. For DAP, both formulations provided similar release onday 92 (301 and 299 μg for formulations C and K, respectively), which iswell in excess of the target minimum level of 200 μg. Release of LNGfrom formulation K was 46 μg on day 92. This is above the lower targetof 35 μg, but less than the higher target of 70 μg. A matrix formulationmay therefore be suitable for use over 3 months.

LNG release is dependent on the initial LNG loading within the ring.Thus, rings with 16 mg LNG release less than those with 32 mg LNG. Thisis apparent for both the levonorgestrel-only rings and the combinationrings (FIG. 5 ).

Levonorgestrel release is significantly increased in the presence of DAPcompared with LNG alone. For example, Day 60 cumulative release for the16 mg LNG-only ring was 885 mcg; when 100, 150 and 200 mg of DAP isincluded in the ring, Day 60 cumulative release was 5004, 5144 and 5408mcg, respectively. Similarly, Day 60 cumulative release for the 32 mgLNG-only ring (red squares, FIG. 5 ) was 2506 mcg; when 100, 150 and 200mg of DAP is included in the ring, Day 60 cumulative release wasincreased to 10934, 11556 and 11611 mcg, respectively. It is clear fromthese data that the actual loading of dapivirine is not important. Basedon the drug loadings tested, it appears that the dapivirine merely needsto be present in the ring in order to enhance levonorgestrel release

Dapivirine in vitro release is highly dependent on DAP loading. FIG. 5Bshows three distinct groups of release profiles (excluding the 25 mg DAPring profile), corresponding to rings containing 100, 150, 200 mg DAP.The LNG component in the rings does not influence DAP release in anymeaningful way.

It is particularly surprising that the same increase in LNG release isobserved in the presence of DAP irrespective of the DAP loading amount.The same increase in LNG would likely be observed when the DAP loadingis reduced to 25 mg, or even 10 mg.

When solid drugs are incorporated into silicone elastomer vaginal rings,a fraction of the solid drug component dissolves in the siliconeelastomer matrix. Assuming that excess solid drug is present in thematrix, the drug will ultimately saturate the silicone elastomer, in thesame way that adding solid drug to water will result in a saturatedsolution. Therefore, for the 100, 150 and 200 mg DAP rings, each wouldcontain the same concentration of dissolved DAP in the siliconeelastomer matrix, despite the differences in overall DAP loading. It isthis fixed concentration, solubilised DAP component in the combinationrings that produces an increase in the LNG release, compared withLNG-only rings. In other words, the dissolved DAP component in thesilicone elastomer is creating an environment in which LNG has increasedsolubility compared with its normal solubility in silicone elastomer.

Freezing-point depression describes the process in which adding a soluteto a solvent produces a decrease in the freezing point of the solvent.Everyday examples include salt in water, alcohol in water, or the mixingof two solids such as impurities in a finely powdered drug. In suchcases, the added compound is the solute, and the original solid can bethought of as the solvent. The resulting solution or solid-solid mixturehas a lower freezing point (melting point) than the pure solvent orsolid did. A eutectic system is a mixture of chemical compounds (orelements) that has a single chemical composition that solidifies at alower temperature than any other composition made up of the sameingredients. This composition is known as the eutectic composition andthe temperature at which it solidifies is known as the eutectictemperature. On a phase diagram the intersection of the eutectictemperature and the eutectic composition gives the eutectic point. Thisphenomenon is also demonstrated for the dapivirine-maraviroc system (seeExample 6, below). The dapivirine-levonorgestrel system also behaves inthe same way, such that the melting point of each component is reducedin the presence of the other component (in a concentration-dependentmanner) such that a eutectic composition exists. Reducing the meltingpoint of a drug in a two-component mixture often correlates with anincrease in its solubility, which in turn enhances permeation andrelease. The increased levonorgestrel release observed from the ringsalso containing dapivirine is likely the result of decreased meltingpoint of the levonorgestrel.

Example 2: Reservoir Rings Comprising a Contraceptive in the Core andDapivirine in the Matrix Sheath

The purpose of this work was to study a combination ring strategycomprising the contraceptive hormone levonorgestrel (LN) in the core anddapivirine in the sheath. Initial work involved the manufacture and invitro release testing of two sets of rings—one having a tin-catalysedsilicone elastomer core (MED8-6382) and the other a platinum-catalysedsilicone elastomer core (DDU-4320). For both formulations, cores wereovermolded with platinum-catalysed silicone elastomer (DDU-4320).

Levonorgestrel was selected as the contraceptive hormone for this study,since initial experiments demonstrated that it is compatible withplatinum-catalyzed silicone elastomer and can be effectively released invitro.

Rings were manufactured using the DDU-4320 grade of platinum-catalysedsilicone elastomer. DDU-4320 is the fully approved medical grade ofLSR9-9508-30.

In an earlier report, a LN release rate of approximately 30 μg/day wasachieved from reservoir rings consisting of a levonorgestrel (LN)-loadedcore with a blank sheath, with both components manufactured fromLSR9-9508-30. However, initial results (reservoir ring with LN-loadedcore and dapivirine (DAP)-loaded sheath, manufactured from DDU-4320)indicated that the LN release was substantially higher than thatpreviously achieved, with a release rate of 124 μg/day. Given themarkedly different release rates, additional rings were manufactured tofurther investigate the increased LN release rate. A second set ofreservoir rings with a LN-loaded core and DAP-loaded sheath,manufactured using DDU-4320, were included in a second in vitro releasestudy. Additional control rings were also manufactured using DDU-4320,and included a LN-loaded matrix ring, a LN-loaded reservoir ring (LN inthe core, blank sheath) and a DAP-loaded reservoir ring (blank core,DAP-loaded sheath).

Dapivirine (micronised) was supplied by IPM. Levonorgestrel(non-micronised) was supplied by Haorui Pharma-Chem Inc. (Irvine,Calif.). DDU-4320 and MED8-6382 silicone elastomers were supplied byNuSil. HPLC-grade acetonitrile, HPLC-grade isopropanol and potassiumdihydrogen orthophosphate (AnalR analytical reagent) were purchased fromVWR International Ltd. (Dublin, Ireland). Phosphoric acid (85% w/w inwater) was purchased from Sigma-Aldrich (Gillingham, UK). A MilliporeDirect-Q 3 UV Ultrapure Water System (Watford, UK) was used to obtainHPLC-grade water.

Reservoir-type vaginal rings consisting of an active core (1% w/w LN)with a DAP loaded sheath (0.3125% w/w) were manufactured, using eitherDDU-4320 (addition cure system) or MED8-6382 (condensation cure system)for the core and (in all cases) DDU-4320 for the sheath, on alaboratory-scale injection-moulding machine. The manufacture ofreservoir rings using this equipment is a three-stage process.

LN-loaded DDU-4320 cores were prepared by mixing 0.5 g LN with 49.5 gsilicone elastomer (part A or part B) in a FlackTek SpeedMixer™ DAC 150FVZ-K (3000 rpm, 1.5 min). Premix A and premix B were combined byspeedmixing (1:1, 3000 rpm, 15 s) and then transferred to a SEMCO®injection cartridge. The cartridge was placed in a manual injection gunand the silicone elastomer was injected into a heated core-mould mountedon the injection-moulder and cured (90° C., 30 s). The dimensions of thecore mould were as follows: Outer diameter=54.9 mm, Internaldiameter=45.9 mm, Cross-sectional diameter=4.5 mm.

MED8-6382 cores were manufactured using MED8-6382 base and MED8-6382crosslinker, which were first blended (25:1, 3500 rpm, 3 min) in aFlackTek SpeedMixer™ DAC 150 FVZ-K. The appropriate quantity of LN wasmixed with the silicone elastomer using the SpeedMixer (3500 rpm, 3 min)to obtain 100 g drug-loaded premix (1% w/w LN). Catalyst (tin(II)2-ethylhexanoate) was added (0.5% w/w) and the formulation wasthoroughly mixed by hand. This active mix was transferred to a SEMCO®injection cartridge, which was placed in a manual injection gun. Coreswere manufactured by injecting the mix into a heated mould (85° C.)mounted on a laboratory-scale injection moulding machine and curing for90 s. A single cut was made in each MED8-6382 core prior to overmouldingto ensure a tight fit in the mould, since shrinkage of the full coreafter curing resulted in misalignment of cores within the sheath when nocut was made.

Once cured, each core was overmoulded in two steps using DAP-loadedDDU-4320 (0.3125% w/w DAP). DAP (0.156 g) was mixed with siliconeelastomer (49.877 g of part A or part B) in the SpeedMixer (3000 rpm,1.5 min). The active premixes (A and B) were combined (1:1) using theSpeedMixer (3000 rpm, 15 s), transferred to a SEMCO® injectioncartridge, injected into a heated mould holding the unsheathed orpartially sheathed core, and then cured (90° C., 90 s). The dimensionsof the full-size ring moulds were as follows: Outer diameter=58.0 mm,Internal diameter=42.8 mm, Cross-sectional diameter=7.6 mm. The sheaththickness of the final rings was 1.55 mm. Six replicates of eachformulation were manufactured and included in in vitro release testing.Prior to the release study, rings were stored at room temperature forthree days in an open container. Rings with a DDU-4320 core will bereferred to as “Set A” throughout this report, while rings with aMED8-6382 core will be referred to as “Set B”.

Following this initial in vitro release study, an additional set ofreservoir rings consisting of a DDU-4320 core (containing 1% w/w LN) anda DDU-4320 sheath (containing 0.3125% w/w DAP) were manufactured and asecond in vitro release study was conducted. The purpose of this secondrelease study was to confirm the results obtained from the first study.This additional ring set will be referred to as “Set C” (sameformulation as Set A but manufactured at a later date). Followingmanufacture, rings in Set C were stored for one week in sealed foilpouches, prior to commencement of the in vitro release study. Duringmanufacture of Set C, cores were cut prior to overmolding in the samemanner as Set B (note that the cores in rings of Set A were NOT cutprior to overmolding). This additional step of cutting the core wasadded to the manufacturing process following the observation that corescomposed of MED8-6382 shrank after curing. Although this was notobserved to the same extent with DDU-4320 cores, it was decided that thecores of Set C should be cut to investigate whether this had any impacton the release of dapivirine and/or levonorgestrel from the rings.

In vitro release testing was performed for all rings over 30 days. Eachring was placed into a 250 mL glass bottle containing 50 mL dissolutionmedium (1:1 mixture of isopropanol and water) and stored in an orbitalshaking incubator (37° C., 60 rpm, 25 mm orbital throw). The dissolutionmedium was sampled and replaced (50 mL) daily with the exception ofweekends (50 mL volume over weekend). Drug release was quantified byreverse-phase HPLC with UV detection.

Details of manufactured rings are presented in Tables 7 and 8.

TABLE 7 Formulation Details for Each Ring Set Ring Set Details AReservoir Ring LN-loaded DDU-4320 core (1% w/w LN) (not cut prior toovermoulding) DAP-loaded DDU-4320 sheath (0.3125% w/w) (first batch) BReservoir Ring LN-loaded DDU-6382 core (1% w/w LN) (cut prior toovermoulding) DAP-loaded DDU-4320 sheath (0.3125% w/w) (only one batchmanufactured) C Reservoir Ring LN-loaded DDU-4320 core (1% w/w LN) (cutprior to overmoulding) DAP-loaded DDU-4320 sheath (0.3125% w/w) (secondbatch)

TABLE 8 Details of rings included in in vitro release study (n = 6).Mean theoretical Mean theoretical Ring Mean ring Mean core Mean sheathLN loading dapivirine loading Set mass (g) (±sd) mass (g) (±sd) mass (g)(±sd) (mg) (±sd) (mg) (±sd) A 7.43 (±0.02) 2.54 (±0)    4.89 (±0.02)25.4 (±0)   15.3 (±0.1) B 7.48 (±0.06) 2.55 (±0)    4.93 (±0.06) 25.5(±0)   15.4 (±0.2) C 7.39 (±0.02) 2.51 (±0.03) 4.88 (±0.02) 25.1 (±0.3)15.3 (±0.1)

Daily and cumulative release rates for DAP and LNG from each ring setare presented in FIGS. 6 and 7 , respectively. A summary of the in vitrorelease data is presented in FIGS. 8 and 9 .

DAP release followed t1/2 kinetics for all ring sets (i.e., Sets A, Band C), as indicated by the release profiles and the linear relationshipbetween cumulative release and root time (R2≥0.995, see FIG. 6 ). Therelease rates for DAP from ring Sets A and C were very similar; 65 and64% of the total DAP loading was released over the 30-day duration ofthe in vitro release study from Set A and Set C, respectively. Theoverall release rates were 1739 μg/day (Set A) and 1703 μg/day (Set C).These two sets of rings were prepared according to an identicalmanufacturing method, with one exception—the cores in ring Set A werenot cut prior to overmolding, while the cores in Set C were cut as ameans of ensuring the core was located centrally within the sheath.Given the similar release profile of DAP from the two formulations, itcan be concluded that cutting the core does not have a marked effect onthe release rate of DAP. The very small decrease in DAP releaseassociated with Set C may be attributed to the reduced length/surfacearea of the core exposed to the overmolded, rate-limiting sheath layer.This indicates that the presence of LN in the core does not have asignificant effect on the release of DAP from the ring.

The release of DAP from Set B was increased compared with Sets A and C.The cores in Set B rings were manufactured from MED8-6382, i.e.,tin-catalysed silicone elastomer. A total of 73% of the initial DAPloading was released over 30 days, at a rate of 1971 μg/day. A likelyreason for the enhanced release from MED8-6382 is the presence ofpropanol in the ring at manufacture, which is produced as a by-productduring curing. The propanol is expected to increase solubility of DAPwithin the rings, and subsequently increase its release rate.

Release of LN followed zero order kinetics (i.e., near constant dailyrelease rates) for all three formulations (R2≥0.9993 for Sets A, B andC). Sets A and C provided similar LN release rates; for both sets, 14%of the original loading of LN was released over 30 days, with a meanrelease rate of 124 and 118 μg/day for Sets A and C, respectively.However, these results are markedly different to those obtainedpreviously for a comparable formulation manufactured from LSR9-9508-30.The rings were manufactured using LSR9-9508-30 and consisted of aLN-loaded core and blank sheath; only 3% of the initial LN loading wasreleased by day 30, with a mean release rate of 30 μg/day.

This discrepancy in the results prompted the manufacture and in vitrorelease testing of Set C, along with additional sets of control rings.Results from Set C confirmed those obtained for Set A, indicating thatthe presence of DAP in the sheath and/or the change in siliconeelastomer batch has caused a substantial increase in the release rate ofLN. LN release from rings consisting of a LN-loaded DDU-4320 core andblank DDU-4320 sheath was also higher than from those rings describedpreviously. This indicates that while factors such as the change in thebatch of silicone elastomer (from LSR9-9508-30 to DDU-4320; samematerial, updated product code) have impacted the LN release rate, thepresence of DAP in the sheath is also causing an increase in LN release.

The release of LN was higher with Set B compared with Sets A and C,following the same trend observed for DAP release; 18% of the totalloading was released at a rate of 159 μg/day. The presence of DAP in thesheath and/or the use of DDU-4320 causes an increase in the release rateof LN.

Summary of Example 2

DAP release follows t1/2 kinetics (typical of matrix devices) and occursat a similar rate regardless of whether LN is present in the core of thering.

LN release follows zero order kinetics (typical of reservoir devices)and occurs at a higher rate when DAP is present in the sheath.

Release of both DAP and LN is increased when the core of the ring ismanufactured from MED8-6382 (tin-catalysed silicone elastomer) ratherthan DDU-4320 (platinum-catalysed silicone elastomer). This isattributed to the solubility enhancing effect of the propanol producedas a by-product of the silicone curing reaction.A sheath thickness of 1.55 mm provides release of LN at a rate ofapproximately 120 μg/day (DAP being present in the sheath).

Example 3: Reservoir-Type Rings Comprising Dapivirine and aContraceptive Loaded into a Single Core

Reservoir-type vaginal rings consist of a drug-loaded core overmouldedwith a rate-limiting polymeric sheath. Drug release occurs via thediffusion of solubilized drug molecules through the core and sheathmaterials followed by drug partitioning into the fluid mediumsurrounding the ring. With excess solid drug in the core to maintain theconcentration of dissolved drug at saturation, drug release fromreservoir-type rings obeys zero order kinetics, i.e., release occurs ata constant daily rate. The rate of drug release depends on the sheaththickness (reducing the thickness of the sheath results in an increasein drug release rate, when release is diffusion controlled), core length(reducing the length of the core results in a decrease in drug releaserate) and the ability of the drug to permeate the sheath (a function ofthe drug's solubility and diffusion co-efficient in the material of thesheath). Unlike matrix-type rings, the drug loading in a reservoir-typering has no effect on the rate of drug release (assuming excess soliddrug is present, as stated above); however, drug loading does influencethe duration of release.

Reservoir rings have the potential to deliver drug(s) over very longtime periods, up to a year and perhaps even longer (assuming there is asufficient quantity of drug located in the core to sustain drug releasefor this length of time). In this study, reservoir-type rings areexplored for the purpose of providing release of both dapivirine (DAP)and levonorgestrel (LNG) for 6 months or longer at constant daily rates.

The simplest reservoir ring design comprises both drugs loaded into asingle full-length core. A disadvantage with this approach may arisewhen the release rate of one drug needs to be altered relative to theother, since changing the sheath thickness and/or core length effectsrelease of both drugs equally. An alternative approach is to load eachdrug into separate cores. In this study, both ring designs areinvestigated—a full-length core ring formulation containing both DAP andLNG, and another ring containing two half-length cores (DAP in the firstand LNG in the second). Release data for the latter ring configurationallowed determination of the individual release rates of both drugs.This information may be used to design a reservoir ring where therelease rate of each drug can be tailored independently of the other inorder to obtain individual target release rates.

Dapivirine (micronised) was supplied by IPM. Levonorgestrel(non-micronised) was supplied by Haorui Pharma-Chem Inc. (Irvine,Calif.). DDU-4320 silicone elastomer was supplied by NuSil.Specifications of each API and the silicone elastomer are provided inAnnex C. HPLC-grade acetonitrile, HPLC-grade isopropanol and potassiumdihydrogen orthophosphate (AnalR analytical reagent) were purchased fromVWR International Ltd. (Dublin, Ireland). Phosphoric acid (85% w/w inwater) was purchased from Sigma-Aldrich (Gillingham, UK). A MilliporeDirect-Q 3 UV Ultrapure Water System (Watford, UK) was used to obtainHPLC-grade water.

Reservoir-type vaginal rings comprising a full-length core containingboth DAP and LNG (each at a loading of 2% w/w) and overmoulded with ablank sheath were manufactured using DDU-4320 on a laboratory-scaleinjection-moulding machine. The manufacture of reservoir rings usingthis equipment is a three-stage process: 1) Injection-moulding of thedrug-loaded core, 2) Overmoulding one side of core with sheath layer, 3)Overmoulding of remaining exposed side of core to produce completelyovermoulded/sheathed core, i.e., reservoir ring. The final ring consistsof a central silicone elastomer core surrounded by a silicone elastomersheath of uniform thickness (FIG. 10 ).

Cores were manufactured by mixing DDU-4320 (part A or B) with theappropriate mass of DAP and LNG (2% w/w loading of both drugs), using aFlackTek SpeedMixer™ DAC 150 FVZ-K (3000 rpm, 1.5 min), to form apremix. Premix A and Premix B were combined by speedmixing (1:1, 3000rpm, 15 s) and then transferred to a SEMCO® injection cartridge. Thecartridge was placed in a manual injection gun and the drug-loadedsilicone elastomer was injected into a heated mould (90° C.) mounted onthe injection-moulder and cured for 30 s. The dimensions of the coremould were as follows: Outer diameter=54.9 mm, Internal diameter=45.9mm, Cross-sectional diameter=4.5 mm. Once cured, a cut was made at asingle point in the core. The core was then transferred to a new mouldset consisting of a lower core mould and an upper fullsize ring mouldfor the first stage of overmoulding (step 2 of the overall process).Parts A and B of DDU-4320 were mixed (1:1) in the SpeedMixer (3000 rpm,15 s), then injected into the heated mould (90° C.) and cured for 90 s,as described for the cores (FIG. 2B).

The third and final manufacturing step involved transferring thepartially sheathed core to another new mould set, consisting offull-size ring moulds on both the upper and lower platform of theinjection-moulder. Overmoulding was completed by injection of the blankDDU-4320 A/B mix prepared for the first overmoulding stage, under thesame conditions (90° C., 90 s cure time) (FIG. 2C). The dimensions ofthe full-size ring moulds were as follows: Outer diameter=58.0 mm,Internal diameter=42.8 mm, Cross-sectional diameter=7.6 mm.

This process produced reservoir rings with full-length cores, loadedwith DAP and LNG (each at a loading of 2% w/w) and overmoulded with ablank sheath. A second formulation consisting of two half-length coresovermoulded with a blank sheath was also manufactured. Each half-lengthcore was loaded with a single drug: one with DAP (2% w/w) and the otherwith LNG (2% w/w), i.e., each drug was formulated separately within thecore of the ring. The two different core formulations were manufacturedfollowing the same process outlined previously for the DAP/LNGcombination core, i.e., full-length complete cores were producedinitially. These cores were cut in half to obtain a half-lengthdrug-loaded core. At step 2 of the manufacturing process, onehalf-length DAP core and one half-length LNG core were each placed inthe mould for overmoulding with blank silicone elastomer. The finalovermoulding step, i.e., step 3 of the process, was the same as thatoutlined for the combination-core reservoir rings.

The two different reservoir-type rings manufactured will be referred toas Configuration 1 and Configuration 2 throughout this report (see FIG.10 ):

-   -   Configuration 1: Full-length core loaded with both DAP and LNG,        each at a loading of 2% w/w, overmoulded with blank DDU-4320.    -   Configuration 2: Two half-length cores, one loaded with DAP only        (2% w/w) and the other loaded with LNG only (2% w/w),        overmoulded with blank DDU-4320.

In vitro release testing was performed daily for 30 days. The samplinginterval was then reduced to twice weekly (on two consecutive days)until day 95, and twice fortnightly thereafter (again, on twoconsecutive days). On Day 0, each ring was placed into a 250 mL glassbottle containing 50 mL dissolution medium (1:1 mixture of isopropanoland water) and stored in an orbital shaking incubator (37° C., 60 rpm,25 mm orbital throw). The dissolution medium was sampled and replaced(50 mL) daily with the exception of weekends (100 mL added to maintainsink conditions).

After Day 30, 50 mL release medium was added over a daily interval and200 mL was added over a weekly/fortnightly interval, as appropriate.Drug release was quantified by reverse-phase HPLC with UV detection.

Details of the rings used in the study are provided in FIG. 10 . Meandaily and cumulative release charts are presented in FIGS. 11 and 12 . Asummary of all in vitro release data is presented in Tables 9 and 10.

TABLE 9 Summary of dapivirine release data for each ring configuration(n = 6) DAPIVIRINE: Configuration 1 Configuration 2 Daily release (μg)(±sd): Day 1 131 (±3)  61 (±20) Day 30 103 (±1)  56 (±1) Day 60 100 (±2) 54 (±1) Day 95  88 (±1)  49 (±1) Cumulative release after 95 days (μg)(±sd) 9670 (±61) 5299 (±51) % Release after 95 days 19% 21% Dailyrelease rate (μg/day) (R²)*    99 (0.9990)    55 (0.9995) Predictedduration of release** ~8.5 months ~7.7 months *linear regressionanalysis was performed when release data was plotted as cumulativerelease against time from day 7 onwards (charts not shown), R² valuerelates to this linear regression. **based on release rate determinedfrom linear regression analysis of cumulative release against time, andassuming that zero order release occurs up to the point of 50% of theoriginal drug lading released.

TABLE 10 Summary of levonorgestrel release data for each ringconfiguration (n = 6) LEVONORGESTREL Configuration 1 Configuration 2Daily release (μg) (±sd): Day 1  8 (±1)  0 (±0) Day 30 138 (±2) 57 (±1) Day 60 134 (±2) 55 (±1)  Day 95 110 (±3) 47 (±1)  Cumulative releaseafter 95 days (μg) (±sd) 12194 (±72)  4792 (± 85)  Daily release rate(μg/day) (R²)*    132 (0.9995)   53 (1.000) % Release after 95 days 24%19% Predicted duration of release** ~6.5 months ~8 months *linearregression analysis was performed when release data was plotted ascumulative release against time from day 7 onwards (charts not shown),R² value relates to this linear regression. **based on release ratedetermined from linear regression analysis of cumulative release againsttime, and assuming that zero order release occurs up to the point of 50%of the original drug loading released.

DAP and LNG release from both ring configurations occurred at a nearconstant daily rate (zero order release) over the 95-day period (FIGS.11 and 12 ), as is typical of reservoir-type devices. These releaserates are expected to continue out to 6 months, or perhaps longer(Tables 9 and 10).

In order to confirm zero order release kinetics, linear regressionanalysis was applied to the cumulative release data from days 7 to 95(release data from the initial timepoints was excluded owing to the lageffect with LNG, FIG. 12A). Linear relationships were observed betweencumulative release and time for all four sets of release data (i.e., DAPand LNG from the two configurations) (Tables 9 and 10).

Release of DAP from Configuration 1 (99 μg/day, full-length core) was1.8 fold higher that from Configuration 2 (55 μg/day, half-length core).Cranks equation predicts a doubling in the release rate, since releaserate is directly proportional to core length.

The LNG release rate increased 2.5 fold when the core length wasincreased from half-length (Configuration 2, release rate 53 μg/day) tofull-length (Configuration 1, release rate 132 μg/day).

In addition to the change in core length, the other difference betweenConfigurations 1 and 2 relates to the formulation of drugs within thecore. In Configuration 1, DAP and LNG are formulated in the samefull-length core of the ring. In Configuration 2, each drug isformulated in separate half-length cores. The results indicate that DAPrelease is not substantially affected by the presence of LNG in the samecompartment, since the release rate appears to be more closelyproportional to core length.

This further indicates that formulation of DAP and LNG in the same coredoes not influence DAP release (note the similar release rate despitethe lower drug loading; release rates are not influenced by loading inreservoir rings, but a higher loaded ring will provide a longer durationof release). However, in the case of LNG, the presence of DAP in thecore may partly explain the greater than 2-fold increase in LNG releasewhen the core length is doubled. Also, the increased surface area fordrug release contributed by the core ends may also be a factor. Finally,it is possible that release of LNG is being influenced by the formationof a eutectic mixture between DAP and LNG in the combination core ofConfiguration 1.

LNG release from reservoir-type rings with a blank sheath was in theregion of 89 μg/day; this increased to 118-124 μg/day when DAP wasloaded in the sheath of the ring.

Summary of Example 3

Release is zero order for both drugs from both configurations

Configuration 1: DAP release rate=99 μg/day; LNG release rate=132μg/day, LNG release rate was higher than DAP release rate, DAP releaserate was lower than the target release rate of 200 μg/day, however, therelease rate may be increased by decreasing the sheath thickness. LNGrelease rate was higher than the target release rate of 70 μg/day,however, the release rate may be reduced by increasing the sheaththickness and/or reducing the core length (as demonstrated by theresults obtained for Configuration 2).Configuration 2: DAP release rate=55 μg/day; LNG release rate=53 μg/day,DAP release rate is similar to LNG release rate. DAP release rate isapproximately half the release rate of Configuration 1, i.e., releaserate is halved by halving the core length. The presence of LNG in thecore in Configuration 1 does not appear to affect DAP release. LNGrelease rate is less than half the release rate of Configuration 1,i.e., release is more than halved by halving the core length. Thepresence of DAP in the core affects LNG release.A reservoir-type ring is suitable for providing constant daily releaseof both DAP and LNG.

Example 4: Vaginal Ring Configurations for Simultaneous Delivery ofDapivirine and Levonorgestrel

Unintended pregnancy, HIV, and other sexually transmitted infections(STIs) all pose major reproductive health issues for women worldwide,particularly in developing countries where access to appropriatemedication/contraceptives is limited. In recent years, there has beenrenewed interest in so-called multipurpose prevention technologies(MPTs) that can simultaneously address two or more clinical indicationsaround reproductive health. MPTs may offer a number of advantages,including a reduced overall cost compared to individual products with asingle indication, and potentially increased user adherence.

Intravaginal rings (VRs) are widely acknowledged as a useful technologyplatform for development of MPTs, since they can readily accommodatemultiple drugs and provide long-term controlled release (FIG. 13 ).Single indication VRs are commercially available for use in hormonalcontraception (e.g., NuvaRing®), and estrogen replacement therapy (e.g.,Estring®). However, no combination therapy VRs are available to date.

In order to produce an intravaginal ring for the simultaneous controlleddelivery of dapivirine (DAP) and a contraceptive, such aslevonorogestrel (LNG) over 30 to 60 days to prevent both sexualtransmission of HIV and unintended pregnancy, four types of intravaginalrings were developed C1-C4) (FIG. 13 ). The rings were manufactured byinjection molding using medical-grade, addition-cured, siliconeelastomer:

i) Configuration 1 (C1)—Matrix ring; loaded with both DAP (0.3125% w/w)and LNG (0.1, 0.3 01′ 1.0% w/w); FIG. 13A.

ii) Configuration 2 (C2)—Reservoir ring; DAP and LNG formulated within asingle full-length core (both 2% w/w), overmoulded with a blank sheath;FIG. 13B.

iii) Configuration 3 (C3)—Reservoir ring; DAP and LNG formulated withintwo separate half-length cores (both 2% w/w), overmoulded with a blanksheath; FIG. 13C.

iv) Configuration 4 (C4)—Reservoir ring; LNG formulated in a full-lengthcore (1% w/w), overmoulded with a DAP-loaded sheath (0.3125% w/w); FIG.13D.

In vitro release was assessed over 30 or 60 days. Each vaginal ring (n=6per formulation) was placed in a stoppered bottle containing 200 mL (C1,day 0, decreased to 100 mL from day 1 onwards) or 100 mL (C2, C3 and C4,day 0, decreased to 50 mL from day 1 onwards) of release medium (1:1mixture of isopropanol and water). Bottles were stored in an orbitalshaking incubator (37° C., 60 rpm) and the release medium was sampledand completely replaced on a daily basis. Drug release was quantifiedusing reverse-phase HPLC with UV detection (DAP: 210 nm; LNG: 240 nm). A25 μL aliquot of each sample was injected onto a Thermo Scientific BDSHypersil C18 column (150 mm×4.6 mm, 3 μm particle size) held at 25° C.,and isocratic elution was performed: mobile phase 55% 7.7 mM phosphatebuffer (pH 3.0)/45% HPLC-grade acetonitrile, flow rate 1.2 mL/min, runtime 9 min, DAP and LNG were eluted after 6.2 and 7.7 min, respectively,The cumulative mass of DAP and LNG released from the variousformulations was compared using a one-way ANOVA, followed by post-hocanalysis with the Tukey-Kramer multiple comparisons test whenappropriate. A p value of less than 0.05 was considered significant.

FIGS. 14A-D depict the daily dapivirine release, cumulative dapivirinerelease, daily LNG release, and cumulative LNG release from ring typeC1, which is a matrix-type ring comprising both DAP and LNG. FIGS. 15A-Ddepict the daily dapivirine release, cumulative dapivirine release,daily LNG release, and cumulative LNG release from ring types C2(reservoir-type ring with both DAP and LNG loaded in the core,surrounded by a blank sheath), and C3 (reservoir-type ring with DAP andLNG each loaded in separate half-cores, surrounded by a blank sheath).FIGS. 16A-B depict daily DAP and LNG release and cumulative DAP and LNGrelease, respectively, from ring type C4 which is areservoir-matrix-type ring with LNG loaded in the core and DAP loaded inthe sheath.

DAP and LNG were released from matrix rings (C1) according to root timekinetics, DAP and LNG release both increased significantly with LNGloading (p<0.05), DAP was released from C2 and C3 at a constant dailyrate (i.e., zero order release), since it was formulated in the core ofthese reservoir rings (102 and 56 μg/day respectively, p<0.05). Allreservoir rings (C2, C3 and C4) provided zero order release of LNG (135,53 and 118 μg/day from C2, C3 and C4 respectively, p<0.05). The rate ofrelease of DAP and LNG from reservoir rings increased significantly asthe core length increased from half-length (C3) to full-length (C2). ForC4 (where DAP was formulated in the sheath of the ring, i.e., similar toa matrix-type ring), DAP release followed root time kinetics, but thetotal cumulative release was significantly lower than C1 (p<0.05),indicating that the presence of LNG in the same compartment increasesthe release of DAP.

Example 5: Intravaginal Ring Comprising Dapivirine and an AntimicrobialCompound

A platinum-catalyzed matrix-type silicone ring was created, comprising25 mg dapivirine and 100 mg of an antimicrobial compound, maraviroc(both micronized and non-micronized). Both micronized maraviroc andnon-micronized maraviroc rings were studied (see FIGS. 17 and 18 ). Ascan be seen in FIGS. 19 and 20 , the release rate of maraviroc from thecombination ring was higher than that of intravaginal rings comprisingonly maraviroc, likely due to the formation of a eutectic composition(see Example 6, below). Initial maraviroc release was lower anddapivirine release higher in micronized maraviroc rings compared tonon-micronized maraviroc rings, but this had a limited effect oncumulative release.

Example 6: Eutectic Behavior of Dapivirine:Maraviroc Mixtures

Maraviroc, dapivirine, and their mixtures were characterized and theresults used to generate a phase diagram for the binary system (FIG. 21). The components are found to produce a simple eutectic system. Curvesare based on freezing point depressing of each component due to thepresence of the other component. The intersection is the estimatedeutectic composition (17.4% dapivirine) and temperature (170° C.).

The pure components were analyzed using DSC, hot stage microscopy, TGA,transmission XRPD, and variable-temperature XRPD. Maraviroc in anorthorhombic crystal form was found to melt at 196° C. Micronized andnon-micronized maraviroc had similar phase behavior, as expected.Dapivirine Form I underwent a solid-solid phase transition to Form II at101° C. which subsequently melted at 220° C. Physical mixtures of thecomponents were analyzed using DSC, hot stage microscopy, andvariable-temperature XRPD. DSC was adequate to observe the solid-solidtransformation in the dapivirine fraction and the onset of eutecticmelting at 170° C. as confirmed using optical hot-stage microscopy.

The kinetics of phase transformations was found to be too slow toeffectively use DSC to determine the full phase diagram. Therefore,variable-temperature X-ray powder diffraction (vT XRPD) was used toinvestigate the phase behavior of maraviroc and dapivirine as a functionof temperature and composition. Slow cooling from the isotropic melt wasemployed until crystals nucleated. Substantial supercooling was requiredto initiate nucleation, particularly near the eutectic composition. Slowheating of the nucleated crystals was employed to bracket a point on theliquidus locus. The liquidus is the maximum temperature at whichcrystals can co-exist with the melt in thermodynamic equilibrium.Fitting functions to each of the branches of the liquidus locuspermitted estimation of both the eutectic composition and temperatureand completion of the phase diagram.

Example 7: Eutectic Behavior of Dapivirine:Levonorgestrel Mixtures

Similar to Example 6, above, levonorgestrel, dapivirine, and theirmixtures were also characterized. The system appears to exhibit eutecticbehavior with the eutectic temperature at about 193.5° C.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. An intravaginal ring comprising 10 mg to 800 mg dapivirineand 10 mg to 1600 mg of an antimicrobial compound, wherein less thanabout 7 mg of dapivirine is released in vitro from said ring during aninitial 24 hour period of release, and wherein the release rate of theantimicrobial compound from the intravaginal ring in vitro is increasedas compared to the release rate of the antimicrobial compound from anintravaginal ring comprising the antimicrobial compound withoutdapivirine.
 2. The intravaginal ring of claim 1, wherein the ring is areservoir-type ring comprising a core and a sheath.
 3. The intravaginalring of claim 2, wherein the dapivirine and the antimicrobial compoundare present in the core of the reservoir-type ring, and the sheath isblank.
 4. The intravaginal ring of claim 2, wherein the dapivirine andthe antimicrobial compound are present in two separate half-lengthcores, and the sheath is blank.
 5. The intravaginal ring of claim 2,wherein the antimicrobial compound is present in the core, and thedapivirine is present in the sheath.
 6. The intravaginal ring of claim2, wherein the core is platinum-catalyzed; or wherein the core comprisesa silicone polymer, an ethylene vinyl acetate EVA polymer, or apolyurethane polymer.
 7. The intravaginal ring of claim 1, wherein thering is a matrix-type ring.
 8. The intravaginal ring of claim 7, whereinthe ring is a platinum-catalyzed ring; or wherein the ring comprises asilicone polymer, an ethylene vinyl acetate EVA polymer, or apolyurethane polymer.
 9. The intravaginal ring of claim 1, whereinbetween about 100 and about 700 μg of dapivirine is released in vitroeach day for 23 days after an initial 7 day period of release.
 10. Theintravaginal ring of claim 1, wherein the antimicrobial compound isreleased in vitro at a rate of between about 200 μg per day and about2000 μg per day, between about 400 μg per day and about 4000 μg per day,between about 550 μg per day and about 5500 μg per day, or between about800 μg per day and about 8000 μg per day for 23 days or 53 days after aninitial 7 day period of release.
 11. The intravaginal ring of claim 1,wherein about 100 mg, about 150 mg, about 200 mg, about 10 to about 30mg, about 25 mg, or about 15 mg of dapivirine is present in the ring.12. The intravaginal ring of claim 1, wherein about 100 to about 800 mg,about 100 mg, about 400 mg, about 800 mg, or about 1600 mg, of theantimicrobial compound is present in the ring.
 13. The intravaginal ringof claim 1, wherein the antimicrobial compound is maraviroc, DS003,darunavir, GSK1265744, or BMS-663068.
 14. The intravaginal ring of claim1, wherein release rates are stable following 3 months of storage; 6months of storage; 12 months of storage; and/or 36 months of storage.15. The intravaginal ring of claim 1, wherein the intravaginal ring has(a) an outer diameter of about 58 mm, an internal diameter of about 43mm and a cross-sectional diameter of about 7.6 mm.; or (b) an outerdiameter of about 57 mm and a cross-sectional diameter of about 7.8 mm.16. A method of blocking DNA polymerization by an HIV reversetranscriptase enzyme in a female human, comprising the step of insertingthe intravaginal ring of claim 1 into the vagina of the female human.17. A method of preventing HIV infection in a female human, comprisingthe step of inserting the intravaginal ring of claim 1 into the vaginaof the female human.
 18. A method of treating HIV infection in a femalehuman, comprising the step of inserting the intravaginal ring of claim 1into the vagina of the female human.
 19. An intravaginal ring comprising10 mg to 800 mg dapivirine and 10 mg to 1600 mg of an antimicrobialcompound, wherein less than about 7 mg of dapivirine is released invitro from said ring during an initial 24 hour period of release, andwherein the dapivirine in the intravaginal ring increases the solubilityof the antimicrobial compound as compared to the solubility of theantimicrobial compound in an intravaginal ring comprising theantimicrobial compound without dapivirine.
 20. An intravaginal ringcomprising a eutectic composition comprising 10 mg to 800 mg dapivirineand 10 mg to 1600 mg of an antimicrobial compound, wherein less thanabout 7 mg of dapivirine is released in vitro from said ring during aninitial 24 hour period of release.